The annotated genome of Streptomyces coelicolor A3(2) revealed 18 cytosolic cytochromes P450 (CYPs) with six ferredoxin (fdx) proteins and two soluble ferredoxin reductases (fpr), their putative electron transport proteins. mRNA expression was observed for all 18 CYPs throughout growth and secondary metabolism, from 3 h after spore germination, and all CYP proteins examined also were present. Expression of members of the fdx complement was detected from the same time point, yet both fpr were detected only at 12 h. Six-hour exposure to dimethylbenzanthracene and benzo[a]pyrene xenobiotics resulted in the absence of some CYP mRNAs and expression of a specific fpr, FR2. This finding and the expression pattern during growth suggested that CYP activity may be regulated by availability of specific reductases. To test this proposal, we expressed in Escherichia coli and purified to homogeneity five CYPs: CYP105D5 (involved in xenobiotic metabolism) and CYP154A1, CYP154C1, CYP158A1, and CYP158A2 (putatively involved in secondary metabolism). Also the two soluble fpr (FR2 and FR3) proposed to shuttle electrons by means of fdx were purified, and specific interactions were observed so that FR2 preferentially reduced CYP105D5 (>90% reduction) compared with the other CYPs (>20% reduction), whereas FR3 preferentially reduced the other CYPs (>85% reduction) compared with CYP105D5 (>10%). Furthermore FR2 was shown to efficiently bind CYP105D5 and drive benzo[a]pyrene hydroxylation in contrast to FR3. These data show that control of CYP activity in S. coelicolor A3(2) involves specific interactions with fpr and their availability during the life cycle and, after xenobiotic exposure, represents a unique mechanism for regulating CYP function.