In Europe, Mycobacterium xenopi is a frequently isolated species among opportunist mycobacteria, and represents one of the main agents of pulmonary infection due to non-tuberculous mycobacteria. Conventional identification of mycobacteria is a time-consuming and laborious process. In this study, we propose a rapid and simple method for the identification of M. xenopi using polymerase chain reaction. The amplified product consists of a specific probe, present in all 38 M. xenopi strains tested, which could not be amplified and did not present cross-hybridization with a set of 110 strains belonging to 23 other mycobacterial species. The probe was cloned and sequenced. Comparison with data bases revealed no significant homologies with previously described sequences.