The effects of variations in Barbour-Stoenner-Kelly (BSK) medium on the infectivity and pathogenicity of Borrelia burgdorferi clinical isolates were assessed by retrospective and prospective studies using a murine model of Lyme borreliosis. Thirty of 35 (86%) mice infected with any of six virulent B. burgdorferi clinical isolates grown in a BSK-H medium developed clinically apparent arthritis. By contrast, arthritis was observed in only 25 of 60 (42%) mice inoculated with two of these B. burgdorferi strains grown in a different lot of BSK-H medium (P < 0.001). In a prospective study, mice inoculated with a B. burgdorferi clinical isolate grown in a BSK medium prepared in-house produced significantly greater disease than those injected with the same isolate cultured in BSK-H medium (P < 0.05). The attenuated pathogenicity is not due to the loss of plasmids during in vitro cultivation. The data suggest that variations in BSK medium have a significant impact on the infectivity and pathogenicity of B. burgdorferi clinical isolates.