Holins represent phage encoded lysis functions required for transit of the phage murein hydrolases to the periplasm. The Lambda S, phage 21 S, and P22 13 holin genes contain a dual translational start motif, beginning with Met1-Lys2-X-Met3. In all cases both start codons at the 5' end of the respective holin gene are utilized. The resulting polypeptides have opposing functions, with the longer product acting as an inhibitor of the shorter one. The 131-codon gene 14 of Bacillus subtilis phage phi 29 encodes the holin function, whereas the downstream gene 15 codes for a lysozyme. phi 29 Gene 14 begins with Met1-Lys2-Met3. Here, we present in vitro and in vivo evidence for the expression of two protein 14 species consisting of 129 and 131 amino acids, respectively. These data suggest that the lysis control mechanism based on two holin species, which has been shown to be operational in the temperature Escherichia coli phages Lambda and 21, and in the Salmonella typhimurium phage P22, is evolutionarily conserved in the lytic B. subtilis phage phi 29.