Previously, we've described a site-directed triple mutant of cytochrome P450 BM3 (BM3) that is able to convert various drugs (van Vugt-Lussenburg, B. M. A., et al. Biochem. Biophys. Res. Commun. 2006, 346, 810-818). In the present study, random mutagenesis was used to improve the activity of this mutant. With three generations of error-prone PCR, mutants were obtained with 200-fold increased turnover toward drug substrates dextromethorphan and 3,4-methylenedioxymethylamphetamine. The initial activities of these mutants were up to 90-fold higher than that of human P450 2D6. These highly active drug metabolizing enzymes have great potential for biotechnology. Using sequencing analysis, the mutations responsible for the increase in activity were determined. The mutations that had the greatest effects on the activity were F81I, E267V, and particularly L86I, which is not located in the active site. Computer modeling studies were used to rationalize the effects of the mutations. This study shows that random mutagenesis can be used to identify novel critical residues, and to increase our insight into P450s.