The nucleotide sequence of a 1.1-kb DNA fragment upstream of a peptidoglycan hydrolase-encoding gene (lytA) from bacteriophage phi 11 of Staphylococcus aureus was determined to see if the upstream sequences are involved in the transfer of the lytA product through the cytoplasmic membrane. Sequencing revealed three open reading frames of 171, 147 and 435 bp with consensus Shine-Dalgarno sequences located upstream from the ATG start codons. The third open reading frame overlaps with the 5' end of lytA by 18 nucleotides. Comparison of the deduced amino acid sequences of the open reading frames with the amino acid sequences in the NCBI Entrez database did not show any significant homology to any sequenced polypeptides. However, the analysis of the peptides showed some structural similarities to the product of the holin gene family. Lysogens containing an insertional mutation in ORF3, upon induction, produced either no phage titer or very low phage titers, compared to the wild-type lysogen. Transformation of ORF3 mutated lysogens by a plasmid containing the intact ORF3 produced the same phage titer as wild-type lysogen, suggesting that the ORF3 product is involved in the process of cell lysis/phage release.