The antimicrobial susceptibility of Borrelia burgdorferi isolated from human spinal fluid was determined in vitro and in vivo. A broth dilution technique was used to determine the MBCs of four antimicrobial agents. The Lyme disease spirochete was most susceptible to ceftriaxone (MBC, 0.04 microgram/ml) and erythromycin (MBC, 0.05 microgram/ml), then tetracycline (MBC, 0.8 microgram/ml), and finally penicillin G (MBC, 6.4 micrograms/ml). Syrian hamsters were used to determine the 50% curative doses (CD50s) of the four antimicrobial agents. Ceftriaxone and tetracycline had the highest activities, with CD50s of 24.0 and 28.7 mg/kg [corrected], respectively. Both erythromycin and penicillin G possessed low activities. The CD50 of erythromycin was 235.3 mg/kg [corrected], and the CD50 of penicillin G was greater than 197.5 mg/kg [corrected].