N-acetyl-L-histidine (NAH) is a major constituent of poikilotherm brain, eye, heart, and muscle, but for which there is no known function. NAH is characterized by high tissue concentrations, a high tissue/extracellular fluid (ECF) gradient, and by a continuous selective and regulated efflux into ECF. In the eye, there is a complete compartmentalization of the synthetic and hydrolytic enzymes, with synthesis of NAH from AcCoA and L-histidine (His) occurring in the lens, and its hydrolysis to acetate and His restricted to surrounding ocular fluids. Using 14C-isotopes, the cycling of NAH between lens and ocular fluids in a simple support medium consisting of NaCl (0.9%), Ca2+ (4 mEq/L) and D-glucose (5 mM) at pH 7.4 has previously been observed. In the present study, using the isolated lens of the goldfish eye, each of the components of that support medium has been individually varied in order to determine its effect on NAH release down its intercompartmental gradient. As a result of these and related studies, it is suggested that NAH may function as a metabolically recyclable gradient-driven molecular water pump. It is proposed that water influx or generation of metabolic water serves as the trigger mechanism to open a Ca-dependent gate for the release of NAH down its gradient, along with its associated water. Preliminary analyses suggest that in addition to its potential for multiple daily cycles, a strongly ionized hydrophilic molecule, such as NAH, may include a large power function as a result of its attraction to water, and it has been calculated that an aqua complex of each NAH molecule may have 33 dipole-dipole-associated water molecules as it passes into ECF. It is this unique combination of a capacity for multiple cycles per day, coupled with a large power function, that may allow for such an intracellular osmolyte to be present in relatively low concentration in comparison to total cellular osmolality, and yet to perform a large and important task with little expenditure of energy. With each NAH molecule recycled up to 10 times/d, and a power factor of 33, there could be 330 mmol of water transported/mmol of NAH each day. With typical NAH concentrations in brains of poikilothermic vertebrates of 5-10 mmol/kg, there is the potential for up to 3.3 mol (60 mL) of water to be removed each day/kg of brain, a value that represents about 8% of total brain water content. Dewatering of the released osmolyte would occur in two additional steps, consisting of its hydrolysis and the subsequent active uptake of its metabolites. It is also suggested that NAH is the archetype of several metabolically and structurally related cellular osmolytes found in both poikilotherms and homeotherms, for which there is similarly no known function, and these may form a family of cycling hydrophilic osmolytes that serve as molecular water pumps in a variety of tissues. These include the basic His containing derivatives: NAH, carnosine, anserine, ophidine, and homocarnosine, and the acidic aspartate derivatives: N-acetyl-L-aspartate (NAA) and N-acetyl-L-aspartylglutamate (NAAG). In each of these cases, the high intracellular/extracellular osmolyte gradient appears to be maintained by combining a hydrophilic protein amino acid with a nonprotein moiety to block its use in other intracellular metabolic pathways, and by blocking catabolism of the derivative by maintaining its hydrolytic enzyme in an extracytosolic membrane or extracellular compartment. Unlike other known water-regulating mechanisms, the proposed cellular system is unique in that as a water pump, it can function as a water regulator independently of extracellular solute composition or osmolality. Finally, based on the hypothesis developed, the NAH system would represent the first cellular water pump to be identified.