Recombinant and native forms of cyclohexanone monooxygenase (CMO) from Acinetobacter NCIB 9871 were analyzed by mass spectrometry to probe ambiguities arising from the presence of multiple DNA sequences for the enzyme in GenBank. A CMO gene corresponding exactly to the nucleotide sequence described by Iwaki ct al. (10) was amplified from genomic DNA, cloned into pET15b, and the recombinant protein purified from a bacterial expression system. Electrospray mass spectrometry of both the recombinant material and the native form of CMO isolated from Acinetobacter yielded molecular weights within 0.01% of those predicted from the translated gene sequence of Iwaki ct al. (10), Trypsin and chymotrypsin digests of native CMO, analyzed by electrospray and MALDI mass spectrometry, provided greater than 97% coverage of the protein and confirmed the presence of specific peptide sequences predicted by the Iwaki sequence alone. Therefore, the primary sequence of native Acinetobacter CMO is identical to the gene sequence for chnB deposited under accession number AB006902.