A previously described mouse peritonitis model was used to study derivatives of Enterococcus faecalis strain OG1RF. The addition of sterile rat fecal extracts (SRFE) lowered the LD50 of OG1RF >10-fold. Hemolysin production caused a 35-fold lower LD50 and a much shorter survival, similar to previous results using a peritonitis model without SRFE. A purine (but not a pyrimidine) auxotroph was considerably less lethal than wild type; gelatinase mutants were also attenuated. A suicide vector was generated with an enterococcal selectable marker in order to disrupt a gene encoding an E. faecalis antigen; the resulting mutant was not attenuated despite a slower growth rate. In conclusion, this model allows attenuated mutants to be detected, corroborates prior reports that hemolysin is a virulence factor, and suggests a role for gelatinase in virulence of E. faecalis in mice; the attenuated purine auxotroph may provide a system for developing vectors for in vivo expression systems.