The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999−36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (V_max = 0.71 nmol/min/nmol, n = 2.18, and S₅₀ = 132 μM). α-Naphthoflavone (α-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S₅₀ value for α-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor.