Double-barreled H(+)-selective microelectrodes were used to study the effect on intravenous acetazolamide on intraretinal pH in the cat. Acetazolamide (11.4-27.8 mg kg-1 intravenously) caused a rapid acidification of the subretinal space. This change in pH originated in the most distal portion of the subretinal space and could not be attributed to a change in pH or PCO2 of the arterial blood. Slow light-evoked alkalinizations in distal retina, attributable to a decrease in rod photoreceptor energy metabolism, were relatively unaltered by acetazolamide. This result indicated that acetazolamide had not crossed the blood-retinal barrier in sufficient amounts to change this response. In time, following intravenous perfusion of acetazolamide, continuous depth profiles of intraretinal pH showed an acidification of the entire retina and the vitreous also became more acidic. These results indicate that the rapid or primary effect of acetazolamide is an acidification of the distal portion of the subretinal space, which is thought to originate in a change in the transport of H+ or HCO3- by the retinal pigment epithelium. This is followed by an acidification of the entire retina and vitreous, presumably due to diffusion of acid from the distal retina, although there could be additional causes.