The objective of this randomized, double-masked, cross-over study was to compare the cardiovascular effects of two glaucoma formulations, ophthalmic 0.5% timolol aqueous solution and 0.1% timolol hydrogel. Twenty-four young healthy subjects received for 2 weeks either twice daily 0.5% timolol solution or once daily 0.1% timolol hydrogel. Heart rate (HR), blood pressure, atrio-ventricular conduction (PR interval), corrected QT time (QTc) and heart rate variability (HRV) were measured in supine position and during head-up tilted position. The mean peak concentrations of timolol in plasma were significantly higher after administration of 0.5% aqueous solution than after 0.1% hydrogel. A 0.5% timolol aqueous solution decreased HR on average by 3 bpm in supine position and by 7 bpm in head-up tilted position while no significant effects were observed with 0.1% timolol hydrogel. During tilt test HR was significantly lower after administration of timolol aqueous solution than after timolol hydrogel (mean +/- SD, 77 +/- 11 bpm versus 86 +/- 13 bpm, P < 0.05). Timolol aqueous solution slightly decreased QTc during tilt (5.9 +/- 5.6 ms, P < 0.01). During tilt tests, timolol aqueous solution slightly increased atrio-ventricular conduction (7.2 ms, P = 0.02). No significant differences were found in HRV. These results indicate that in healthy volunteers, ophthalmic 0.5% timolol aqueous solution produces more pronounced cardiac beta-blocking effects than 0.1% timolol hydrogel.