In 1994, we isolated Borrelia burgdorferi sensu lato from 231 patients with erythema migrans who presented to the University Medical Center in Ljubljana, Slovenia. Samples of erythema migrans-affected skin were placed in media to support the growth of Borrelia species and evaluated in Ljubljana and Chicago. Patients whose cultures were positive included 132 women and 99 men; 136 of these 231 patients recalled a tick bite. Patients noted a rash an average of 24 days after a bite and presented a mean of 34 days after the bite with erythema migrans (mean diameter. 16 cm). Itching (44%) burning (18%), and pain (11%) were the most common local symptoms. Systemic complaints (40%) included headache, fatigue, malaise, and arthralgia. Other than erythema migrans, findings on physical examination were minimal (< 5% had fever, and in < 10% local lymph nodes were affected). Serial serological studies using indirect immunofluorescence assay, ELISA, and Western blot methods were performed, and antibodies to B, burgdorferi sensu lato were detected in < 50% of samples from patients. This is the largest series reported to date of patients with culture-confirmed Lyme borreliosis. It highlights the deficiencies of serological tests in early disease, demonstrates the sensitivity of direct detection methods for evaluation of patients with erythema migrans, and suggests that patients with early Lyme borreliosis in Slovenia may suffer a milder illness than those in the United States.