In a double-masked, randomized, placebo-controlled study, we evaluated the effect of topical timolol maleate 0.5% on the retinal, choroidal, and optic nerve head circulation in 5 healthy volunteer subjects. Changes in the pulsatile component of choroidal blood flow (PCBF) were determined from measurements of the ocular pulse wave. Changes in the retinal arterial blood flow rate (RBF) and optic nerve head capillary blood speed (CBS) were determined by laser Doppler velocimetry and monochromatic photography. In timolol-treated eyes, PCBF decreased by 32 ± 12% (p = 0.0007). Changes in RBF and CBS were not statistically significant. In the contralateral placebo-treated eyes, PCBF decreased by 15 ± 8% (p = 0.006) and RBF increased by 18 ± 10% (p = 0.002). The change in CBS was not statistically significant.