A pyrimidine analogue, 5-fluorouracil (5-FU), was injected intravitreally into one eye of the goldfish, either alone, or before or after injection of the same eye with the dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA). Effects of these agents were explored by measuring their actions on numbers of proliferating cell nuclear antigen-immunoreactive (PCNA-ir) cells, representing mitotically active rod precursor cells (RPCs) in normal conditions, in both treated and untreated retinas. At various intervals (5–45 days after the first injection of drug), the retinas ( n = 6 for each treatment group) were isolated and processed as wholemounts by an indirect immunohistochemical method for PCNA. Some retinas were cryosectioned and processed for certain immunoreactive cells other than PCNA. Changes in the mean density of PCNA-ir cells, following intravitreal 5-FU (10 μg/2 μl saline on 3 consecutive days) alone or in combination with intravitreal 6-OHDA (2.5 μg/2 μl), were statistically compared for interval vs. day 0 (control from intact retinas) and for treated vs. contralateral (untreated or treated) retinas, in both outer and inner nuclear layers (ONL and INL, respectively). 5-FU at this dose drastically reduced the densities of endogenous and 6-OHDA-induced PCNA-ir cells in the ONL of treated retinas, but transiently increased them in the contralateral untreated retinas, probably reflecting an injury influence from the treated retina. The density of PCNA-ir cells at the retinal margin was also greatly reduced in treated retinas. Such changes peaked on days 10–15, and gradually disappeared over days 30–45. In the INL, on the other hand, 5-FU did not cause a significant change of the PCNA-ir cell density in either treated or untreated retinas. As expected, 5-FU appeared not to act upon retinal cells immunoreactive to certain phenotype-specific antibodies other than PCNA. The results indicate that 5-FU strongly prevents proliferation of RPCs in the ONL and of neuroblasts at the retinal margin, but negligibly influences unidentified PCNA-ir cells in inner retina. They showed also that clustering of PCNA-ir cells and the marked increase of their density, seen in 6-OHDA-intoxicated retinas, are due to vigorous proliferation of neuroblast cells in the fish retina.