Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, D,L-3-hydroxybutyrate, acetoacetate, alpha-oxoisohexanoate and alpha-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor alpha-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.