In vitro studies have demonstrated direct interactions between Borrelia burgdorferi and human B and T cells. However, largely because disseminated infections typically occur at very low density, little is known about associations between spirochetes and mammalian host cells in vivo. To assess whether spirochetes interact directly with lymphocytes in mammals, we developed a mouse model for lymphotropism. By repeatedly coincubating spirochetes with primary mouse lymphocytes that were immobilized by adherence to immunomagnetic beads, we were able to preferentially enrich cultures for or against bacteria with constitutive affinity for murine B and T cells. Populations of lymphotropically enriched, stock infectious, and lymphotropically depleted spirochetes were injected intradermally into mice. Lymphocytes were then purified from the blood and spleens of challenged mice and placed into spirochetal culture medium. Cultures of B. burgdorferi were obtained from primary lymphocyte preparations from mice challenged with each of the three populations of spirochetes. Recovery of lymphocyte-associated bacteria occurred within 1 h of challenge with enriched bacteria. Lymphocyte preparations from mice challenged with stock infectious and lymphotropically depleted bacteria produced cultures after 1 day postchallenge. All lymphocyte preparations were culture negative after 1 week. These results demonstrate that lymphotropic B. burgdorferi is infectious in mice and suggest that associations between spirochetes and lymphocytes occur in vivo. The results also suggest that factors involved in lymphocytic binding may be inducible in vivo. Thus, this system provides a model for studying the role of such interactions in mammalian infections.