Nebivolol hydrochloride (R067555), is a new antihypertensive drug. Aromatic and alicyclic hydroxylation at the benzopyran ring systems of nebivolol are important metabolic pathways. Generally, NMR is used to unambiguously assign the sites of hydroxylation. Because of the low dose rates and the extensive metabolism of nebivolol in the different species, NMR identification is not always possible, and therefore another spectroscopic technique was searched for to address this problem. UV-chromophore absorption is affected by the kind and arrangement of adjacent atoms and groups (auxochromes). The effect of these auxochromes (e.g. -NH2, -NR2, -SH, -OH, -OR and halogens) can be strongly influenced by the pH. This paper proves that HPLC at high pH combined with on-line diode-array detection is an excellent technique for the location of the hydroxyl functions in hydroxylated metabolites of nebivolol. With this technique it is possible to differentiate between glucuronidation at the automatic and aliphatic or alicyclic hydroxyl functions.