Tolerance to penicillin exists among the viridans group of streptococci, but its therapeutic significance is unknown. We studied the effect of penicillin alone and in combination with streptomycin, in vivo and in vitro, on three strains of dextran-producing Streptococcus sanguis serotype II which possess widely various degrees of penicillin tolerance. In rabbits with experimental endocarditis, treatment with procaine penicillin (250 mg/kg intramuscularly twice daily for 5 days) decreased the number of viable organisms in valvular vegetations from 8.82 log10 +/- 0.98 CFU/g in untreated controls to 5.31 +/- 1.19 for a highly tolerant strain, 4.22 +/- 1.05 for a less tolerant strain, and 1.79 +/- 1.72 for a nontolerant strain (P less than or equal to 0.01 for comparison between any of the four groups). None of 36 rabbits infected with tolerant strains were cured by 5 days of treatment with penicillin, but 10 of 23 animals infected with the nontolerant strain were cured (P = 0.00002). When streptomycin was given in combination with penicillin, rabbits infected with the nontolerant strain were cured within 3 days, and rabbits infected with the tolerant strain were cured within 5 days. These findings indicate that tolerance can exert a critical influence on the response of S. sanguis to penicillin therapy in vivo and that the combination of penicillin plus streptomycin exerts a synergistic effect against tolerant as well as nontolerant organisms.