7-Hydroxycoumarin (7-HC) has been used as a probe substrate for phase II enzymes uridine 5'-diphospho-glucuronosyltransferase (UGT) and sulfotransferase (ST) in both microsomal and in cellular systems. It may be used directly as a substrate to measure phase II activities or may be the intermediate metabolite from the phase I metabolism of the parent substrates 7-ethoxycoumarin or couarmin. In humans, 7-HC glucuronide is the predominant phase II metabolite. However, the specificity of 7-HC as a phase II substrate for the UGT isozymes has not been reported. To investigate 7-HC specificity as a substrate for UGT isozymes, we used a panel of recombinant human enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). Rank order of the activity showed a stratification amongst the UGTs tested: (1A6, 1A9) > (1A1, 1A10, 2B7, 2B15) > (1A3, 1A4, 2B4, 2B17). These groups spanned a wide range of activities from UGT1A6 at 3808 pmol/min/mg, to UGT1A1 at 289 pmol/min/mg to UGT1A3 at 31 pmol/min/mg. Km and Vmax determination was performed to confirm the rank order. The results show that 7-HC is not metabolized equally between the UGT isozymes. This data may aid in interpreting metabolism differences between tissues or donors due to differential distribution of UGT isozymes.