Following single drop instillation of pilocarpine, drug levels in individual aqueous humor samples of rabbits were determined by high-performance liquid chromatography and correlated with changes in pupillary diameter and intraocular pressure (IOP). Application of higher concentrations of pilocarpine resulted in higher drug levels but these differences were neither statistically significant nor consistently proportional to the strength applied. Maximum miosis occurred between 30 and 60 minutes and the maximum fall in IOP occurred between 1 and 2 hours. The influence of contact lenses with varying water content and thickness upon intraocular penetration of pilocarpine was studied. Contact lenses did not affect 30-minute concentrations of pilocarpine in aqueous humor. Although two-hour levels were significantly lower with contact lenses, miosis and IOP at these time periods were comparable with and without lenses. Neither water content nor thickness appreciably influenced miosis and IOP.