To determine the intraocular concentrations of Mitomycin C (MMC) after extraocular application, we used pigmented rabbits and placed sponges soaked with MMC under the conjunctiva on top of the intact episclera. First, we soaked the sponges with volumes ranging from 0.025 ml to 0.3 ml of the solution containing MMC and sampled aqueous humour after 30 minutes. The concentrations, as determined by high performance liquid chromatography, did not correlate to the amount of MMC given. Then, we soaked the sponges with different concentrations of MMC with a volume of 0.1 ml and sampled aqueous humour, vitreous humour and sclera after 60 minutes. The concentrations of MMC were higher in the vitreous than in the aqueous, and, relative to these values, very high in the sclera. These results indicate that the amount of MMC reaching the interior of the eye after standard extraocular application may be highly variable, and that the concentrations of MMC in the sclera and formed vitreous can be considerably higher than in the aqueous humour.