P450 enzymes have attracted the attention of chemists for decades because of their impressive ability to catalyze the hydroxylation of inactivated C--H bonds. However, their use for synthesis in aqueous systems is limited. We report here a survey of the activity of purified human CYP3A4 in the presence of organic solvents or ionic liquids. We show that CYP3A4 tolerates only small amounts (<15 %) of water-miscible organic cosolvents or ionic liquids before its activity toward testosterone drops below detection. [BMIM][PF(6)] in a biphasic system was less detrimental to enzyme activity, with 20 % of the activity remaining in the presence of 15 % of this ionic liquid. CYP3A4 activity in the absence of buffer was only >or=10 % in solvents of the alkane series, with a minimum of 0.85 % water, and with the addition of sucrose and testosterone before enzyme lyophilization. Biphasic solvent systems were more promising, with approximately 85 % of the activity retained.