Because mitomycin C reduces the resistance to aqueous outflow normally provided by postoperative subconjunctival fibrosis, we would expect to see more frequent hypotony after trabeculectomy with mitomycin C than after standard trabeculectomy. To evaluate the incidence of hypotony in trabeculectomy with intraoperative mitomycin C use, we performed a retrospective analysis on 52 eyes of 48 patients who underwent trabeculectomy with mitomycin C. Mitomycin C concentration was 0.4 mg/ml in all eyes, and treatment time ranged from 3 1/2 to seven minutes. We defined hypotony as intraocular pressure lower than 5 mm Hg on two examinations at least four weeks apart and six weeks or more postoperatively. Overall, average intraocular pressure was 22.7 +/- 10.7 mm Hg preoperatively and 10.4 +/- 5.0 mm Hg postoperatively (P < .001), a mean reduction in intraocular pressure of 12.3 +/- 11.5 mm Hg (47.0%). Hypotony occurred in 17 of 52 eyes (32.7%). Seven eyes required trabeculectomy revision for hypotony. Hypotonous eyes received longer treatment with mitomycin C intraoperatively, with a mean application time of 5.3 +/- 1.0 minutes for hypotonous eyes and 4.7 +/- 0.8 minutes for nonhypotonous eyes (P = .03). Sixteen of 43 eyes (37.2%) undergoing primary filtration became hypotonous, as compared to one of nine (11.1%) eyes that had previous filtering procedures (chi 2 = 2.30, P = .13). Nine of 17 hypotonous eyes (52.7%) and five of 35 nonhypotonous eyes (14.3%) had loss of two or more lines of Snellen visual acuity. Hypotony occurred in nearly one third of eyes treated with mitomycin C during trabeculectomy in our study. There was a statistically significant (P = .03) association of hypotony with longer application time of mitomycin C, and a trend toward increased incidence of hypotony in primary filtration.