The authors investigated effects of two antimetabolites, 5-fluorouracil (5-FU) and mitomycin C (MMC), on proliferation and motility of cultured rabbit subconjunctival fibroblasts. Both drugs caused dose-dependent inhibition of fibroblast proliferation. These effects were reversible at lower doses (0.33 mg/l of 5-FU, 1.0 X 10(-3) mg/l of MMC) and at an early stage of treatment, but became irreversible at higher doses or after longer treatment times. The degree of growth inhibition reached a plateau after 7 days of treatment. Neither drug altered fibroblast migration in the agarose droplet motility assay or on the intra-cytoplasmic actin distribution by epifluorescent-light microscopy. These results indicate that 5-FU and MMC inhibit subconjunctival fibroblast function predominantly by their effect on cell proliferation rather than on cell motility. The clinical implications of these results may possibly support the use of a much lower total dose of subconjunctival antimetabolite than used in the 5-FU clinical trial.