A phage-associated lysin was found in culture lysates resulting from the propagation of virulent bacteriophage A25 on the group A streptococcal strain designated K56. In contrast to the previously described group C streptococcal phage-associated lysins, A25 phage-associated lysin was more active on chloroform-treated cells, was not phage bound, and was active on some group G and H strains, as well as on group A and C strains. A25 phage-associated lysin had an optimum pH of 6.7 and was inactivated by 10(-3) M p-hydroxymercuribenzoate. Group A cells exposed to penicillin were more susceptible to A25 phage-associated lysin, whereas chloramphenicol-treated cells became resistant to lysis. Release of lipoteichoic acid appeared to precede lysis, and cardiolipin treatment of cells reversed the effects of chloroform and penicillin treatments. These results suggest the possibility that A25 phage-associated lysin may have a mechanism similar to the mechanism of an autolysin or that cell lysis may be due to the activation of an autolysin.