Eighty-eight strains of group B streptococci were studied to determine their in vitro susceptibility to penicillin and rifampin alone and in combination. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined by broth microdilution; studies of synergy were performed by microdilution checkerboards and time-kill curves. The MIC90 and MBC90 for penicillin were both less than or equal to 0.06 microgram/ml. The MIC90 and MBC90 for rifampin were less than or equal to 0.25 microgram/ml and greater than 32.0 microgram/ml, respectively. Checkerboard MIC determinations revealed synergy for 46 strains (52%), an additive or indifferent effect for 42 strains (48%), and no antagonism. MBC checkerboards showed antagonism for 62 strains (70%); in 45 (73%) of these 62 strains, high concentrations of rifampin increased the MBC of penicillin, whereas low concentrations decreased the MBC of penicillin. Time-kill studies performed on selected strains supported the results of MBC checkerboards, although concentrations of antibiotics that were antagonistic in the checkerboards had an indifferent effect in time-kill experiments. When concentrations of antibiotics were used that are clinically achievable in cerebrospinal fluid, delayed killing was seen even though synergy had been observed in both MIC and MBC checkerboards.