Mycobacterium paratuberculosis is indistinguishable from M. avium by 16s rRNA sequence analysis. Consequently, the commercial rRNA probe (AccuProbe for M. avium complex, GenProbe, San Diego, CA) can not distinguish between the two organisms. DNA homology with M. avium is roughly 99%. Phenotypically, M. paratuberculosis is much slower growing, dependent on mycobactin for in vitro cultivation and capable of causing a chronic inflammatory bowel disease in ruminants known as Johne's disease, while M. avium is essentially avirulent for nonimmunocompromised mammals. Mycobacterium paratuberculosis has also been found in human patients with Crohn's disease. Genetically, the only way to distinguish M. paratuberculosis from M. avium is to test for the presence of the insertion element IS900. A PCR-amplified DNA probe for IS900 is commercially available (IDEXX Laboratories, Inc. Westbrook, ME). To determine if HPLC analysis of mycolic acid extracts could identify M. paratuberculosis, over 15 isolates of the organism were subjected to HPLC as previously described for identification of mycobacteria. Initially the computer program could not distinguish the chromatogram of M. paratuberculosis from that of M. intracellulare. However, visual inspection of the HPLC pattern revealed subtle differences. Once the HPLC pattern of M. paratuberculosis was entered into the pattern recognition library, the system successfully distinguished M. paratuberculosis from M. intracellulare and M. avium grown on the same culture medium. HPLC analysis may provide a more rapid and cost-effective means of identifying M. paratuberculosis than use of genetic probes.