Some strains of the coagulase-negative Staphylococcus lugdunensis produce a synergistic hemolytic activity (SLUSH), phenotypically similar to the delta-hemolysin of S. aureus. Reverse-phase high-pressure liquid chromatography of supernatants from S. lugdunensis 307 yielded three late-eluting peaks of 3.5 kDa with synergistic hemolytic activity. A degenerate oligonucleotide probe was designed from partial amino acid sequences of the 23-amino-acid (aa) tryptic fragments from one of the three peaks and hybridized to a single 2.8-kb HindIII chromosomal fragment. The relevant portion of this fragment was cloned by PCR, and sequencing showed the presence of three related open reading frames (ORFs), SLUSH-A, SLUSH-B, and SLUSH-C, preceded by an unrelated short potentially coding sequence (ORF-X), cotranscribed on a polycistronic 838-nucleotide mRNA. The amino acid sequences of the peptides from the three peaks align perfectly with the predicted sequences from the three SLUSH ORFs (peak I = SLUSH-B; peak II = SLUSH-C; peak III = SLUSH-A). These three peptides are closely related (amino acid homology, >76%) and do not show significant homology to S. aureus delta-hemolysin but do resemble a Salmonella typhimurium invasin and the "gonococcal growth inhibitor," a bacteriocin secreted by Staphylococcus haemolyticus. The predicted ORF-X gene product is a 24-aa peptide with no homology to the SLUSH peptides.