The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, alpha- and beta-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the omega(-2) position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids alpha-ionone (similar to 70% conversion) and beta-ionone (similar to 91% conversion) yielded the allylic alcohols 3-hydroxy-alpha-ionone and 4-hydroxy-beta-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.