In an in vitro study, rabbit subconjunctival fibroblasts were cultured and the effects of an antineoplastic drug, hydroxyurea (HU), on fibroblast proliferation and fibroblast attachment was investigated. The effects of HU were compared with those of mitomycin C (MMC). Different concentrations of HU and MMC were added to culture medium. The HU doses which led to 50% of inhibition (ID50) and the dose which led to about 90% of inhibition (subtoxic high dose, STHD) were determined to be 8 and 1,000 μg/ml, respectively. ID50 of MMC and its STHD which led to about 100% inhibition were found to be 0.01 and 1 μg/ml, respectively. Reversibility studies revealed that inhibition disappeared depending on the dose and incubation period of both HU and MMC. In an in vivo study, glaucoma filtration surgery (GFS) was performed in rabbits which were treated with HU (treatment group) and distilled water (control group). Tissue samples were taken from the subconjunctival area treated at 1 h, 1 day, 5 days and 30 days postoperatively. The biopsy specimens were then placed in tissue culture media. Fibroblast outgrowth rates detected in the HU group were found to be significantly lower than those in the control group in the specimens taken at the end of the first hour. The difference was significant on culture days 9–15 in the biopsy specimens taken on day 1 while it was not significant in those taken on days 5 and 30.