Transport of timolol and befunolol in the anterior uvea of the albino rabbit was studied in vitro. Also, the mode of timolol elimination from the living eye was investigated. The isolated iris-ciliary body incubated at 37 degrees C accumulated 14C-timolol and 14C-befunolol against the concentration gradient, and the tissue-to-medium ratio was 5.45 +/- 0.29 and 5.46 +/- 0.44, respectively. The accumulation of both drugs was significantly suppressed by incubation at 0 degrees C and also by pretreatment with ouabain or cyanine but not with probenecid. The relationship between the initial velocities of accumulation and medium drug concentrations conformed with saturation kinetics, indicating the presence of mediated transport of these drugs. The apparent Km was 1.08 X 10(-6) mole per liter for timolol and 8.00 X 10(-7) for befunolol, and the Vmax was 5.46 X 10(-9) mole per g per hour for timolol and 1.52 X 10(-8) for befunolol. A mixture of 14C-timolol and 3H-sucrose was injected into the center of the vitreous cavity of the living rabbit. Timolol was lost from the vitreous at a rapid rate of about 36% per hour, while sucrose was lost very slowly, at about 5% per hour. Thus it was thought that an energy-requiring carrier-mediated transport system is operative in the anterior uvea of the albino rabbit, and that this would account for the rapid removal of the beta-blockers out of the eye.