A bacteriophage specific to Bacillus amyloliquefaciens, a gram-positive bacterium, was isolated from a local sewage treatment center. Using a lysis assay, a gene, lys1521, was isolated and its nucleotide sequence revealed one open reading frame of 375 bp. Homology studies showed amino acid alignment similarity with gene 5A of Bacillus subtilis phages PZA and phi 29. Overexpression of the cloned gene yielded a 13 kDa protein corresponding to the predicted gene product. Despite the fact that no significant homology with known cell wall lytic enzymes was apparent, the lytic profile obtained in an in vivo expression assay showed that lys1521 had cell wall hydrolysis activity. This is a significant revelation since the function of the homologous gene 5A product of phage phi 29 has been suggested to be required for the in vivo elongation of phage DNA replication. The lys1521 gene could be evidence of the presence in gram-positive bacteriophages of a third lysis gene in addition to the well characterized two-step lysis system.