Enterococci are resistant to penicillin killing in vivo and in vitro. Because some bacteria resistant to penicillin killing have reduced autolytic activity, we examined the lysis of clinical enterococcal isolates suspended in buffer (spontaneous lysis), and compared it with their susceptibility to antibiotic-induced lysis and killing. We found significant correlations between spontaneous and antibiotic-induced lysis, using five antibiotics that inhibit cell wall synthesis (penicillin, cephalothin, bacitracin, cycloserine, and vancomycin). Among isolates, strains more rapidly lysed by one antibiotic were more rapidly lysed by the other antibiotics, and more susceptible to spontaneous lysis. In studies involving a single strain grown in different media, spontaneous lysis also correlated closely with antibiotic-induced lysis. These results are consistent with a common mechanism for spontaneous and antibiotic-induced lysis, such as the autolytic enzyme system. Human serum was one of the least permissive media tested for enterococcal growth and antibiotic-induced lysis and killing. We suggest that the inhibitory effect of human serum on growth and the activation of the enterococcal autolytic enzyme system may be a critical factor in the resistance of enterococcal endocarditis to treatment with penicillin alone.