Hemicholinium-3 effects on the albino rabbit neural retina have been described, but effects on the retinal pigment epithelium (RPE) have not been closely examined. We have studied retinal morphology and function in Dutch belted (pigmented) rabbits after single intravitreal injections of Hemicholinium-3 or saline. DC electroretinogram recordings show a decrease in a, b, and c-wave amplitudes, with the c-wave affected first. Experiments with sodium iodate show that the early decrease in the c-wave results from a loss of the RPE component of the c-wave, rather than the retinal Slow PIII component. After two days, ophthalmoscopic abnormalities of the fundus are severe in a large area with pigmentary changes. A sharp boundary appears between normally pigmented and depigmented fundus, indicative of a critical threshold for damage. The RPE contains clumped melanin. Pigmented cells are seen away from the basement membrane, an early histological observation temporally correlated with a loss of barrier function seen in fluorescein angiograms. After 10 days, apparent proliferation of non-pigmented RPE cells coincides with re-establishment of the barrier. Rod photoreceptor outer segments are lost 4–7 days after injection in the depigmented regions of the fundus, but outer segments are spared in normally pigmented fundus areas. This regional pattern is distinct from that seen in albino rabbit retina where outer segment loss is fairly uniform. We conclude that Hemicholinium-3 affects the RPE in pigmented rabbits in addition to known effects on retinal cholinergic neurons and photoreceptor disc synthesis.