Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.