An ear punch biopsy method for the detection and isolation of Borrelia burgdorferi from rodents was developed. The ear punch biopsy proved to be extremely sensitive, detecting spirochetes in 100% (11 of 11) of laboratory hamsters infected by tick bite and 95.8% (23 of 24) of hamsters infected by intraperitoneal inoculation. When cultured at 4 to 6 weeks postinfection, 92 to 100% of the ear punches taken from individual hamsters yielded viable spirochetes. B. burgdorferi was detected in sequential cultures from animals as early as 4 days postinfection and as late as 20 weeks postinfection. A total of 86% (6 of 7) of field-collected white-footed mice (Peromyscus leucopus) which were positive for B. burgdorferi as determined by xenodiagnosis were also positive by the ear punch method. The ear punch biopsy method allows individual rodents to be sampled for B. burgdorferi serially over a long period and thus should prove useful for both field and laboratory experiments.