The adjunctive use of antimetabolites has revolutionized surgical procedures to correct glaucoma in the past decade. Although much is known about the in vitro and in vivo actions of 5-fluorouracil and mitomycin C, their use in the eye has raised new questions about cellular toxicity. For example, filtering blebs after a surgical procedure supplemented with mitomycin C are relatively avascular in comparison with those seen with 5-fluorouracil supplementation. We investigated the effect of 5-fluorouracil and mitomycin C on cultured capillary endothelial cells and fibroblasts. The viability and morphologic characteristics of 3T3 fibroblasts and capillary endothelial cells were studied in light of increasing doses of 5-fluorouracil and mitomycin. Both cell types showed similar sensitivity to mitomycin C. However, fibroblasts were far more sensitive to 5-fluorouracil than were the cultured microvascular endothelial cells. Thus 5-fluorouracil appears to be toxic to fibroblasts while sparing vascular endothelial cells, whereas mitomycin C is cytotoxic for both cell types.