Previously, two genes, designated as lyt and hol, were identified in the lysis module of phage mu1/6. They were cloned and expressed in Escherichia coli. An additional candidate holin gene, hol2, was found downstream from the hol gene based on one predicted transmembrane domain and a highly charged C-terminal sequence of the encoded protein. Expression of hol or hol2 in E. coli was shown to cause cell death. The concomitant expression of lambda endolysin (R) and mu1/6 holin resulted in cell lysis. Similarly, the coexpression of the endolysin and holin of phage mu1/6 led to lysis, apparently due to the ability of mu1/6 endolysin to hydrolyze the peptidoglycan layer of this bacterium. In contrast, the simultaneous expression of mu1/6 hol2 and the endolysin gene (lambdaR or mu1/6 lyt) did not cause detectable lysis of the host cells. Demonstration of the holin function in streptomycetes was achieved by providing for the release of mu1/6 endolysin to the periplasm and subsequent cleavage of the peptidoglycan, which strongly suggested that the holin produces lesions in the streptomycete membrane.