A number of antimicrobial agents have been used in the treatment of human brucellosis with varying effectiveness. The purpose of this study was to test the in vitro susceptibility of isolates of four Brucella species to a variety of antimicrobial agents, and to study in vitro synergy of combinations of agents. Minimal inhibitory concentrations (MICs) were determined using conventional broth microdilution methods and commercially available systems. Conventional checkerboard synergy microdilutions were prepared for gentamicin or streptomycin plus tetracycline, and rifampicin plus tetracycline. Synergy or antagonism was determined by the fractional inhibitory concentration index. Penicillin G and ampicillin showed in vitro activity against Brucella (MIC90 4 micrograms/ml), whereas the antipseudomonal penicillins were less active (carbenicillin MIC90 12 micrograms/ml, piperacillin MIC90 32 micrograms/ml). Among the third generation cephalosporins tested, cefotaxime (MIC90 2 micrograms/ml) demonstrated greatest activity. As a class, aminoglycosides were equivalent (MIC90 1-4 micrograms/ml). All strains were sensitive to tetracycline (MIC90 0.25 microgram/ml), trimethoprim-sulfamethoxazole (MIC90 1/19 micrograms/ml), and rifampin (MIC90 1 microgram/ml). Erythromycin (MIC90 greater than 8 micrograms/ml) and vancomycin (MIC90 greater than 16 micrograms/ml) demonstrated no activity. In vitro synergy (fractional inhibitory concentration index less than 0.5) was demonstrated with tetracycline plus rafampin in six of eight isolates tested.