Although penicillin tolerance has been increasingly recognized among clinical isolates of many Gram-positive organisms, the significance of this phenomenon in vivo is not clear. The present study was performed to characterize penicillin-tolerant enterococci by several in-vitro parameters and to examine the significance in vivo in a rabbit model of infective endocarditis. Tolerant enterococci exhibited several characteristics which distinguished them from non-tolerant bacteria: significantly greater ratios of MIC to MBC of penicillin, resistance to penicillin-induced lysis and killing, and growth in areas of superinhibitory concentrations of penicillin upon transfer from penicillin gradient to penicillin-free plates. In-vivo studies of aortic valve endocarditis in rabbits treated with procaine penicillin G (300 mg/kg/day) revealed strikingly different responses between infections due to one tolerant and one non-tolerant strain. Animals infected with a tolerant enterococcus showed consistently greater bacterial counts in vegetations during ten days of therapy and significantly lower rates of vegetation sterilization. Serum penicillin levels were not significantly different between the two groups, but serum bactericidal titres were significantly lower for the tolerant than for the non-tolerant strains. These findings indicate that penicillin tolerance identified by several in-vitro criteria is a significant determinant of the in-vivo response of enterococci to penicillin therapy.