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Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysidqtotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenferiae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tubernlosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium awium and Mycobacterium bowis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium waccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His,-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bowis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitm.