Barbour-Stoenner-Kelly (BSK) II medium containing fosfomycin, 5-fluorouracil, trimethoprim and sulfamethoxazole was evaluated for the selective isolation of the Borrelia species responsible for Lyme disease and relapsing fever. The maximum non-inhibitory concentrations of fosfomycin, 5-fluorouracil, trimethoprim and sulfamethoxazole for six strains of borreliae were 500 to > 1000 micrograms/ml, 250 to > 500 micrograms/ml, 125 to 500 micrograms/ml and 125 to 500 micrograms/ml, respectively. The combination of four agents (fosfomycin 400 micrograms/ml, 5-fluorouracil 100 micrograms/ml, trimethoprim 10 micrograms/ml, sulfamethoxazole 50 micrograms/ml) did not inhibit the growth of borreliae, allowing growth in cultures inoculated with a few organisms (theoretically a single organism). In contrast, the four-agent combination completely inhibited the growth of 12 of 13 other bacterial strains tested as possible contaminants. This combination also allowed the selective growth of borreliae in experimentally contaminated specimens. The four-agent combination in BSK II medium may be useful for selective isolation of Borrelia species responsible for Lyme disease and relapsing fever from clinical and environmental samples.