Ceftriaxone is highly effective clinically in patients with Lyme disease. We studied a representative invasive human isolate of Borrelia burgdorferi for which the MBC of ceftriaxone was 0.050 μg/ml. A once-per-day dosage regimen of ceftriaxone (50 mg/kg/dose) administered intramuscularly for 5 days was 100% effective in sterilizing tissue samples of C3H mice infected with this strain of B. burgdorferi, regardless of whether the mice were being treated concomitantly with a corticosteroid. Administration of the same five doses of ceftriaxone at 6-h intervals over just 24 h was also 100% effective. These experiments suggest that shorter courses of antibiotics than those currently recommended should be considered for study in patients with early uncomplicated Lyme disease.