The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.