Sorbitol and mannitol, two stereoisomeric osmolytes, inhibit the ATP-dependent Ca2+ transport in inside-out vesicles derived from basolateral membranes from kidney proximal tubules. This inhibition (I0.5 = 400 and 390 mM respectively) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by these osmolytes. In the presence of 1 M sorbitol or mannitol, Ca2+ uptake is inhibited by 70 and 75%, respectively. Since the Ca(2+)-stimulated ATPase activity is unaffected, sorbitol and mannitol uncouple the Ca2+ transport from the ATPase activity. The inhibition of Ca2+ transport by these osmolytes is reversible, since the inhibition disappears when the vesicles are preincubated with 1 M sorbitol or mannitol and then diluted 25-fold in reaction medium to measure Ca2+ accumulation. On the other hand, these osmolytes protect the (Ca2+ + Mg2+) ATPase from the inhibition of Ca2+ transport and ATPase activity by urea and guanidinium. These data suggest that the high concentrations of polyols that renal cells accumulate during antidiuresis, may regulate Ca2+ transport across the plasma membrane. In addition, polyols may protect the (Ca2+ + Mg2+) ATPase from the deleterious structural effects of urea, a compound that also accumulates during antidiuresis.