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The effect of topical 0.5% timolol on the microcirculation in albino rabbit optic nerve head (ONH) tissue was examined using a laser speckle microcirculation analyser. In the first experiment, normalized blue (NB) in ONH, a quantitative index of peripheral blood flow in ONH, intraocular pressure (IOP), blood pressure (BP) and pulse rate (PR) were measured under general anesthesia before, and 30, 60, 90 and 120 minutes after a 20 microliters instillation of timolol in one eye and a placebo in the other eye in a masked randomized manner. In the second experiment, one eye of another group of rabbits received timolol twice daily for 20 days and a placebo in the fellow eye in a randomized masked manner. The NB in ONH and IOP were measured before treatment and 2 hours after the last instillation on the 20th day. After a single instillation of timolol, PR showed a maximum reduction of 11% and the IOP in the timolol-treated eyes showed a maximum decrease of 18%, but the NB in ONH and BP did not show any significant change during the experiment. After the 20-day treatment, IOP showed a maximum decrease of 28% in the timolol-treated eyes and 17% in the placebo-treated eyes. The NB in the timlol-treated eyes increased significantly (p0.01), but in the placebo-treated eyes it showed no significant change. Thus, the long-term use of topical timolol may increase peripheral blood flow in ONH tissue in the living rabbit eye.