The purpose of this study was to gather information on the mechanism by which timolol/pilocarpine (TI/PI) combination eyedrops provide additive ocular hypotensive effects. An hypothesis, according to which the combination eyedrops prolong the intraocular permanence of PI as a consequence of decreased aqueous humor secretion induced by TI, was not supported by clear-cut literature evidence. It was thus sought to verify if repeated instillations in albino rabbits of combination TI/PI eyedrops do effectively prolong the turnover of PI. Commercial eyedrops containing 0.68% w/v TI maleate and 2.0% w/v PI hydrochloride, buffered at pH 6.8, and two reference solutions containing PI hydrochloride alone (2% w/v), buffered at pH 5.5 and 6.8, were instilled b.i.d. in albino rabbits for five days. Aqueous humor samples, analyzed after the last treatment, showed that the aqueous humor PI levels observed after administration of the combination eyedrops were significantly higher than those resulting from administration of the reference formulations. When compared with the pH 6.8 reference solution, the pH 5.5 one produced slightly higher and more sustained drug levels in the aqueous humor. The present results appear to confirm the assumption that an increased retention of PI in the aqueous humor is responsible for the additive effects on intraocular pressure reported by several authors for the combination TI/PI eyedrops.