The presence of the membrane transport mechanism, lactate-H+ cotransport, was tested in explants of rabbit corneal epithelium. Basal corneal epithelial cells were loaded with the pH sensitive fluorescent dye BCECF. Intracellular pH (pHi) was measured by rationing the fluorescence emission output following excitation at 490 and 440 nm. Perfusion of explants in lactate-containing Ringer's, pH 7.40, produced a reversible decrease in pHi. The lactate induced proton influx (mM/min) followed saturating kinetics, Km = 10.7 mM lactate, Vmax = 10.2 mM/min. Proton influx following addition of 10 mM lactate was inhibited 36, 60 and 47% by pre-perfusion in 1 mM CHC (cyano-hydroxycinammic acid), 500 microM H2DIDS (4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid) and 1 mM LAIE (lactic acid isobutylester), respectively. These inhibitors of lactate-H+ cotransport were reversible. Mersalyl acid (500 microM) inhibited proton flux from 10 mM lactate addition by nearly 100%, but was irreversible. Stimulation of lactate production by perfusion in N2 equilibrated Ringer's (hypoxia) or the addition of 1 mM NaCN led to a slow alkalinization (0.1 pH unit in 10 min). Pre-perfusion with the reversible inhibitors slowed the hypoxic alkalinization by approximately 40%. It is concluded that lactate-H+ cotransport is present in the corneal epithelium and that it contributes to pHi regulation during hypoxia.