We have studied the effect of retinal illumination on the concentration of the extracellular space marker tetramethylammonium (TMA+) in the dark-adapted cat retina using double-barreled ion-selective microelectrodes. The retina was loaded with TMA+ by a single intravitreal injection. Retinal illumination produced a slow decrease in [TMA+]o, which was maximal in amplitude in the most distal portion of the space surrounding photoreceptors, the subretinal space. The light-evoked decrease in [TMA+]o was considerably slower and of a different overall time course than the light-evoked decrease in [K+]o, also recorded in the subretinal space. [TMA+]o decreased to a peak at 38 s after the onset of illumination, then slowly recovered towards the baseline, and transiently increased following the offset of illumination. It resembled the light-evoked [TMA+]o decreases previously recorded in the in vitro preparations of frog (Huang & Karwoski, 1990, 1992) and chick (Li et al., 1992, 1994) but was considerably larger in amplitude, 22% compared with 7%. As in frog, where it was first recorded, the light-evoked [TMA+]o decrease is considered to originate from a light-evoked increase in the volume of the subretinal space (or subretinal hydration). A mathematical model accounting for [TMA+]o diffusion predicted that the volume increase underlying the response was 63% on average and could be as large as 95% and last for minutes. The estimated volume increase was then used to examine its effect on K+ concentration in the subretinal space. We conclude that a light-dependent hydration of the subretinal space represents a significant physiological event in the intact cat eye, which should affect the organization of the interphotoreceptor matrix, and the concentrations of all ions and metabolites located in the subretinal space.