1. The capability of human CYPs other than 2E1 to catalyse the formation of 6-hydroxychlorzoxazone (6OHCHZ) was examined in vitro using human liver microsomes. 2. 4-Methylpyrazole, diethyldithiocarbamate (DDC), and rabbit anti-human CYP2E1 antibodies reduced chlorzoxazone 6-hydroxylase activity by 60, 60 and 50% respectively. The rate of formation of 6OHCHZ by DDC-treated microsomes was reduced further by the 3A inhibitors midazolam, troleandomycin and gestodene and increased by alpha-naphtholavone, a 3A4 stimulator. 3. Following preincubation with DDC there were significant correlations (p < 0.05) between the residual CHZ 6-hydroxylase activity and immunoquantified CYP3A levels, and corresponding activities (e.g. midazolam 1'-hydroxylation). Rabbit anti-human CYP3A antibodies alone and in combination with DDC reduced the formation of 6OHCHZ by 47 and 62", respectively. 4. cDNA expressed CYP3A4, 2E1 and 2D6 exhibited comparable CHZ 6-hydroxylase activity. CHZ modulated 3A4 activity as reflected by midazolam 1'-hydroxylase and 4-hydroxylase activities. 5. CYP3A may make a significant contribution to CHZ 6-hydroxylation and therefore caution should be exercized when chlorzoxazone is employed as a specific 2E1 probe in vitro and in vivo.