A new isolate, Mycobacterium sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn5-derived mutants, we characterized 2 mutants defective in growth on pristane or n-hexadecane. A single copy of Tn5 was found to be inserted into the coding region of mcr (alpha-methylacyl coenzyme A [alpha-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of mls (malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in Escherichia coli was confirmed to catalyze racemization of (R)-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 micromol . min(-1) . mg of protein(-1). Real-time quantitative reverse transcriptase PCR analyses indicated that mcr gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used (S)-2-methylbutyric acid for growth but did not use the racemic compound, and growth on n-hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the (R) isomer, although the (R) isomer was not toxic during growth on n-hexadecane. Based on these results, Mcr is suggested to play a critical role in beta-oxidation of methyl-branched alkanes in Mycobacterium. On the other hand, mutant H1 could not grow on n-hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through beta-oxidation of pristane.