The locus of insertion of a transposon previously used to obtain a non-haemolytic avirulent mutant was identified: it is the structural gene encoding lisreriolysin O, the thiol-dependent haemolysin, now called hlyA. The gene was completely sequenced. The preliminary structural and functional study of the chromosomal region containing the gene indicates that hlyA belongs to a monocistronic transcriptional unit. If it is the case, the transposon insertion would have no major polar effect on downstream genes and would only affect hlyA expression. These results emphasize the importance of the haemolysin in the virulence of Listeria monocytogenes.