Expression of a gene encoding a novel protein antigen of 40 kDa (p40) was detected in IS901(+) strains of Mycobacterium avium, but not in any other species or subspecies of Mycobacterium tested, including IS901(-) M. avium and the other members of the M. avium complex. Although Southern hybridization revealed that the p40 gene is widely distributed within the genus, expression of the antigen could not be detected on Western blots of mycobacterial cell lysates. Nucleotide sequence analysis of the cloned p40 gene, and a database search, revealed high levels of sequence identity with a homologous gene in IS901(-) M. avium, M. avium subsp. paratuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium smegmatis and Mycobacterium tuberculosis. Further analysis of upstream sequences identified a putative promoter region. The p40 gene is the first example of a gene that is widely distributed within the genus Mycobacterium but expressed only in association with the presence of a genomic insertion element, in this case IS901, in strains of M. avium isolated from birds and domestic livestock.