A new method of evaluating quantitative cytotoxicity in cell culture was demonstrated using Chang's cultured human conjunctival cells. Time-dependent cytotoxicity of some beta-adrenergic blocking agents was shown and expressed by exposure time, causing 50% cell damage (50% cell damage time CDT50). Pure timolol maleate maleate of 0.25% and 0.5% concentration caused no cytotoxicity within a 64-min exposure. Pure befunolol hydrochloride of 0.25%, 0.5% and 1% inhibited 10%-30% of cell growth at 64-min exposure. Pure bupranolol of 0.125%, 0.25%, 0.5% and 1% showed evident cytotoxicity and CDT50 at 66 min 48 s, 38 min 54 s, 3 min 46 s and 1 min 18 s, respectively. Commercial preparations, which contained benzalkonium chloride as a preservative, indicated more rapid cytotoxicity. Timolol preparations of 0.25% and 0.5% showed CDT50 at 48.1 s and 2 min 4 s, respectively. Befunolol preparations of 0.25%, 0.5% and 1% showed CTD50 at 43.4% s, 4 min 38 s and 58 s, respectively, Bupranolol preparations of 0.125%, 0.25%, 0.5% and 1% demonstrated CDT50 at 1 min 11 s, 3 min 24 s, 22.3 s and 13.7 s, respectively.