In rats challenged with viridans streptococci poorly susceptible to antibiotic killing, single doses of antibiotics only prevent endocarditis induced by bacterial inoculum sizes that produce disease in 90% of control animals (ID90): additional doses are required to protect against inocula exceeding the ID90. We investigated whether single-dose rifampin would extend the efficacy of single-dose prophylaxis to inocula exceeding the ID90. We used two strains of viridans streptococci highly susceptible to killing by rifampin and two resistant strains. All rats were injected with 10-1,000 times the ID90 of the four strains. Single-dose rifampin successfully prevented endocarditis due to all four strains. A few prophylaxis failures were observed after challenge with the two poorly susceptible strains, but in vivo emergence of resistant variants did not account for these failures. Thus, rifampin was the first antibiotic given as a single dose that successfully prevented experimental streptococcus endocarditis after challenge with high bacterial inocula.