Mycobacterium malmoense was first described in 1977. It is now recognized as an opportunistic human pathogen which can be difficult to identify using standard methods. M. malmoense may be underestimated as the causative agent of clinical disease because of the recognized difficulties in its primary cultivation and identification. In this study, the nucleotide sequence of the 16S/23S rRNA intergenic spacer region from five clinical isolates of M. malmoense has been determined, in order to develop a PCR-based DNA probe assay to facilitate the early identification of this organism. The DNA sequence generated was utilized to design an oligunucleotide probe that specifically hybridizes with M. malmoense. The ability of this DNA probe to detect geographically distinct M. malmoense isolates was investigated. The value of this DNA probe was realized by its ability to differentiate three isolates of the Mycobacterium avium complex, which had been misidentified as M. malmoense using conventional biochemical methods.