Lyme disease spirochetes were isolated from the kidneys of two Peromyscus spp. trapped in Minnesota in September and October 1983. No spirochetes were isolated from white-tailed deer (Odocoileus virginianus), red backed voles (Clethrionomys gapperi), or shrews (Sorexy cinereus and Blarina brevicauda). This is the first report of the isolation of the Lyme disease spirochete from the midwestern United States and isolations from these animals, which were free of ticks, suggest that the Lyme disease spirochete may persist in animal organs for months.