Twenty clinical isolates of Staphylococcus aureus were examined to determine the frequency of rifampin-resistant variants. All isolates were highly susceptible to rifampin, with mean minimum inhibitory concentrations of 0.11 +/- 0.1 microgram/ml and mean minimum bactericidal concentrations of 0.22 +/- 0.2 microgram/ml. The frequency of isolation of resistant variants was similar to all rifampin concentrations tested. Rifampin-resistant variants maintained their resistance upon daily subculture in rifampin-free broth. Rifampin-susceptible S. aureus exhibited a growth and survival advantage over the rifampin-resistant mutants both in pure cultures and in mixtures with rifampin-resistant antecedents. A comparison of the virulence for mice of five susceptible isolates and their 100 microgram/ml-rifampin-resistant variants showed that two of the resistant variants were less virulent than the susceptible strains via intraperitoneal challenge, whereas three of the resistant variants were less virulent by intravenous challenge (P less than 0.05).