Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized.