1 A method for the assay of debrisoquine 4-hydroxylase activity in vitro by microsomal fractions of human liver is described. The assay utilises gas chromatography-mass spectrometry with d9-4-hydroxydebrisoquine as internal standard. 2 The limit of detection of 4-hydroxydebrisoquine was 2 ng ml -1 and the coefficient of variation was 4.4%. 3 Debrisoquine 4-hydroxylase activity was linear with protein to concentrations above 2.1 mg ml -1 and with incubation times of at least 15 min. 4 Debrisoquine 4-hydroxylase is a microsomal enzyme with a requirement for NADPH. Activity was inhibited by carbon monoxide. It is concluded that the activity is catalysed by cytochrome P-450. 5 In three samples of human liver the mean value for Vmax of debrisoquine 4-hydroxylase activity was 69.9 +/- 14.3 pmol mg -1 min -1 and for Km it was 130 +/- 24 microM. 6 The only variable from smoking status, alcohol ingestion, sex of the patients, source of liver sample and presence of liver disease that had a significant effect on 4-hydroxylation of debrisoquine was the presence of liver disease. This was associated with a decrease in enzyme activity.