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A large number of microsomal P450s have been expressed in Escherichia coli in quantities sufficient for structure/function analysis. However, only one mitochondrial P450 has been successfully overexpressed, that being cholesterol side chain cleavage cytochrome P450 (P450scc). We report here overexpression, purification, and characterization of a second mitochondrial P450, human sterol C-27 hydroxylase (P450c27). The conditions used for expression are very similar to those applied for P450scc, although a quite different purification protocol was necessary to achieve highly purified P450c27. The catalytic properties of purified recombinant human P450c27 resemble those of purified, endogenous rat and rabbit P450c27, regarding both specificity and turnover numbers. Like endogenous P450c27 from rat and rabbit liver, human recombinant P450c27 is only functional in the presence of adrenodoxin and adrenodoxin reductase and shows no activity in the presence of the microsomal P450 reductase. We conclude that P450c27 is most likely not the 1alpha-hydroxylase of 25-hydroxyvitamin D3, contrary to a previous suggestion (Axen, E., Postlind, H., Sjöberg, H., and Wikvall, K. (1994) Proc. Natl. Acad. Sci. USA 91, 10014-10018) because this activity of P450c27 (28 pmol/min/nmol P450) seems far too low to be physiologically relevant. This activity is 10(3) times lower than the 27-hydroxylase activity toward 5beta-cholestane-3alpha,7alpha,12alpha-triol and 40 times lower than the 27-hydroxylation of cholesterol by this enzyme. The development of this expression system and purification procedure creates the potential for structure/function analysis of P450c27, including possible crystallization of this important enzyme.