In enterococci, the structural gene for beta-lactamase (blaZ) is identical to blaZ from Staphylococcus aureus. However, in the enterococci studied to date, beta-lactamase is produced constitutively, whereas in staphylococci it is often inducible. Recent reports have revealed the presence of two adjacent genes upstream of the staphylococcal blaZ thought to be the antirepressor (blaR1) and repressor (blaI) genes. In the present study, beta-lactamase expression mutants of the staphylococcal beta-lactamase plasmid pI524 were generated by transposon mutagenesis with the transposon Tn917. Tn917 insertions upstream of blaZ in either blaR1 or blaI resulted in constitutive beta-lactamase production, indicating that the repressor function is lost with insertion of Tn917 into either gene. This finding supports the concept that the staphylococcal beta-lactamase regulatory genes are encoded on a polycistronic mRNA. The corresponding region upstream of the enterococcal blaZ from Enterococcus faecalis HH22 was sequenced and compared with the staphylococcal blaR1 sequence. The two sequences were identical for 893 nucleotides, and then the sequences diverged completely. Therefore, in strain HH22, only 51% of the putative antirepressor gene is present and the repressor gene is also absent. In conclusion, constitutive beta-lactamase production in HH22 appears to be due to a lack of the regulatory genes blaR1 and blaI which regulate expression of blaZ in staphylococci.