The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450 monooxygenase is important to the growing field of combinatorial biosynthesis. Using the pikAIV deleted mutant of Streptomyces venezuelae HK954, we have shown that PikC could catalyze the hydroxylation of an alternative substrate, oleandomycin. The hydroxylated form of oleandomycin has been detected primarily by thin layer chromatography and high-performance liquid chromatography. We have isolated the cytochrome P450 gene pikC from streptomyces venezuelae which is responsible for the C-12 hydroxylationof narbomycin to pikromycin and PikC was purified for further studies.