Three methods were tested for recovering Borrelia burgdorferi from live mice onto BSK II culture medium. Four laboratory-reared Peromyscus leucopus were inoculated intraperitoneally with the JD-1 isolate of Borrelia burgdorferi. Borrelia burgdorferi spirochetes were recovered from 13 of 20 (65%) samples taken by needle aspiration between days 7 and 40 post-inoculation (PI) and from 1 of 16 samples of skin obtained by ear punch biopsy during the same sampling period. Spirochetes were not recovered from culture media inoculated with mouse blood. The use of needle aspirates for recovering spirochetes was compared among three species of mice: P. leucopus, P. maniculatus, and Reithrodontomys fulvescens. Spirochetes were isolated from 14 of 15 aspiration samples from four P. maniculatus, 12 of 20 from three P. leucopus, and 15 of 20 from four R. fulvescens taken between days 7 and 48 PI. Spirochetes were isolated from only one aspiration sample between days 80 and 95 PI from any of the mice tested. Needle aspiration was an efficient method for repeated recovery of B. burgdorferi from live, experimentally infected mice. We also document R. fulvescens as an experimental host for B. burgdorferi. Based on their susceptibility to infection, all species of mice tested herein may play a role in the epidemiology of Lyme disease where their distribution is compatible with endemic transmission.