Single-doses or short-term administration of beta-lactam antibiotics alone or combined with aminoglucoside antibiotics have failed to consistently prevent experimental streptococcal endocarditis induced by high inocula of bacteria poorly susceptible to killing by these antibiotics. The optimal duration of administration of antibiotics for successful prophylaxis under these circumstances has not been established. We therefore tested, in rats with catheter-induced sterile aortic vegetations, the duration of administration of antibiotic necessary to prevent endocarditis induced by bacterial inocula 100 to 10,000 times the 90% infective dose of two tolerant viridans-group streptococci and two Streptococcus faecalis strains. Multiple-dose regimens of amoxicillin alone or of amoxicillin combined with gentamicin were studied. Against the two viridans group streptococci, successful prophylaxis was achieved with multiple doses of amoxicillin alone given over 24 to 48 hr and by the combination of amoxicillin and gentamicin given for 6 to 24 hr. Against the two S. faecalis strains, multiple-dose regimens with amoxicillin alone failed, but the combination of amoxicillin and gentamicin was successful when administered for 48 to 72 hr. Thus, after challenge with high bacterial inocula, repeated doses of a beta-lactam antibiotic alone were sufficient to prevent viridans streptococcal endocarditis, but multiple doses of a bactericidal combination (beta-lactam plus aminoglucoside), as necessary for the treatment of established endocarditis, were a prerequisite for successful prophylaxis of S. faecalis endocarditis.