Live Borrelia burgdorferi were isolated from the blood and/or urine of white-footed mice (Peromyscus leucopus) collected on Shelter Island, New York, in 1984 and 1985. Prevalence of spirochetes in urine was consistently higher than in blood or both fluids simultaneously. Spirochetes remained viable for 18-24 hours in urine and were maintained in culture for one week. Mice removed from the field were spirocheturic for at least 13 months. One spirocheturic mouse developed spirochetemia one month after field removal indicating the pathogen can return to the peripheral circulation. Twenty-one kidneys from 22 mice had spirochetes in the interstitial areas and bridging the tubules. A positive correlation between Babesia microti infection and spirocheturia was seen. Although the mechanism of entry into the urine is unknown, B. microti infection may increase glomerular permeability. Babesia induced hematuria may provide possible nutrients to maintain spirochetes. Urine may provide a method for contact non-tick transmission of B. burgdorferi in natural rodent populations particularly during periods of nesting and/or breeding.