To determine the ability of environmental bacteria to metabolize the frequently prescribed fluoroquinolone drug ciprofloxacin, eight Mycobacterium spp. cultures were grown for 4 days in a medium containing sorbitol and yeast extract with 100 mg x L(-1) ciprofloxacin. After the cultures had been centrifuged and the supernatants extracted with ethyl acetate, two metabolites were purified by using high-performance liquid chromatography. They were identified with liquid chromatography/electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin was transformed to both N-acetylciprofloxacin (2.5%-5.5% of the total peak area at 280 nm) and N-nitrosociprofloxacin (6.0%-8.0% of the peak area) by Mycobacterium gilvum PYR-GCK and Mycobacterium sp. PYR100 but it was transformed only to N-acetylciprofloxacin by Mycobacterium frederiksbergense FAn9, M. gilvum ATCC 43909, M. gilvum BB1, Mycobacterium smegmatis mc2155, Mycobacterium sp. 7E1B1W, and Mycobacterium sp. RJGII-135. The results suggest that biotransformation may serve as a ciprofloxacin resistance mechanism for these bacteria.