We determined if the ear biopsy location affected detection of Borrelia burgdorferi when either culture or the polymerase chain reaction (PCR) was used among 50 white-footed mice (Peromyscus leucopus), live-captured in a Lyme disease enzootic area in Maryland (USA) between March and October of 1991 and 1992. The infection status of individual mice was determined by organ culture; ear biopsy samples were obtained from the peripheral and central part of the ear for detection of B. burgdorferi by culture and by PCR. Overall, B. burgdorferi was cultured from one or more tissue samples in 33 (66%) of 50 captured mice. Among infected mice, B. burgdorferi was detected by culture in 29 (88%) of 33 peripheral and 28 (85%) of 33 central ear biopsy samples. By PCR it was detected in 24 (73%) of 33 peripheral and all 33 central samples (P = 0.002). Detection of B. burgdorferi by culture was independent of the ear biopsy location; however, the organism was detected by PCR with greater frequency in central ear biopsy samples as compared to peripheral samples. Agreement between culture and PCR was moderate (Kappa = 0.64) on peripheral ear samples and excellent (Kappa = 0.79) on central samples. We propose that when ear biopsy samples are used to detect B. burgdorferi by PCR in wild-caught P. leucopus, removal of biopsy samples from the central part of the ear will achieve maximum sensitivity and will achieve the highest concordance between assays when both culture and PCR of ear biopsy samples are conducted in parallel.