The aim of the study was to determine mitomycin-C (MMC) concentrations in human aqueous humour during trabeculectomy and to correlate anterior chamber concentrations with method of application. MMC was applied intra-operatively by saturating sponges, ranging in size from 2 x 2 x 5 mm to 2 x 4 x 10 mm on dry cut, with 0.5 mg/ml MMC during trabeculectomy for 3-5 minutes. Applications to episclera were made in 18 cases and to the scleral bed after scleral flap dissection in 9 cases. Aqueous samples were collected by paracentesis with a 30 gauge needle 2-7 minutes after removal of sponge and external irrigation. Aqueous MMC concentrations were determined by high-performance liquid chromatography. Aqueous MMC concentration in 27 samples ranged from below minimum detectable concentration (less than 5 ng/ml) to 120.8 ng/ml. Mean aqueous drug levels obtained when the applications were to the scleral bed were 35.65 +/- 39.17 ng/ml (range 5-120.8 ng/ml). Applications on episclera gave mean aqueous concentrations of 4.98 +/- 9.11 ng/ml (range 0-33.3 ng/ml). The difference was statistically significant (p = 0.004). There were no correlations between sponge size, time of MMC exposure and aqueous MMC level. In conclusion, MMC is detectable in aqueous humour within minutes of external application and the aqueous concentration level is higher if the application is in the scleral bed than on the episclera. Toxicity of the drug at this concentration range for corneal endothelial cells needs further investigation via in vitro and clinical studies.