A reversed-phase high-performance liquid chromatographic method is described for the determination of betaxolol in human aqueous humour. Betaxolol and the internal standard metoprolol were extracted with cyclohexane and separated on a reversed-phase column (Luna C-18, 250x4.6 mm, 5 mum) with a mobile phase containing acetonitrile-phosphate buffer (40:60, v/v) at a flow-rate of 0.8 ml/min. The column effluent was monitored with a fluorescence detector at 227 nm (excitation) and 301 nm (emission). The retention times for metoprolol and betaxolol were 3.55 and 5.63 min, respectively. The recovery from aqueous humour was found to be 71.6% for betaxolol at 1.25 mug/ml. The within-day and day-to-day accuracy values were in the range of 96.17-105.2% for betaxolol at 0.1, 4 and 12 mug/ml (n=6), within-day and day-to-day precision values were less than 10% for betaxolol at the concentrations given above, The detection limit corresponding to the signal-to-noise ratio of 3:1 was 15 ng/ml. The presented method was suitable for measuring betaxolol levels in human aqueous humour samples obtained from patients after topical administration. (C) 2002 Elsevier Science B.V. All rights reserved.