The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space. This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2+/-0.1 x 10(-4)cmsec(-1) (osmoll(-1))(-1). In solutions containing 50 mmoll(-1) lactate, however, the mannitol-induced cell shrinkage was faster and the cells alkalinized. These effects were not linear functions of the magnitude of the imposed osmotic gradients: Both volume effects and changes in intracellular pH showed apparent saturation with increasing gradients. Abrupt isosmotic replacement of Cl(-) with lactate in the concentration range from 3 to 50 mmoll(-1) caused an immediate cell swelling as well as an immediate intracellular acidification; both effects showed apparent saturation with increasing lactate concentration. The K(m) values were: 11+/-2 mmoll(-1) for the water fluxes and 13+/-4 mmoll(-1) for the H(+) and lactate fluxes. The data suggest that H(2)O is cotransported along with H(+) and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue and supports our previous study performed by an invasive technique in an amphibian tissue.