Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, floxuridine, and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications.