A total of 1,019 Amblyomma, americanum, 479 Dermacentor variabilis, 6 Ixodes texanus, and 1 I. dentatus ticks was collected from mammals and vegetation on three sites in Indiana, from March to October, 1986. Midgut diverticula were examined for the etiological agent, Borrelia burgdorferi, of Lyme disease (LD), using dark-field and immunofluorescence microscopy, and tissue samples cultured in BSK II medium for 7 to 21 days. Spirochetes were isolated in the BSK II medium from pools of midgut diverticula from 6 D. variabilis from Martin County and pools of midgut diverticula from 7 D. variabilis from Lawrence County. Spirochetes from Martin County differed morphologically from B. burgdorferi, and did not react positively to the rabbit anti-B. burgdorferi conjugate from the Montana NIH Laboratories. Spirochetes from Lawrence County were morphologically similar to B. burgdorferi, and reacted positively to direct immunofluorescence tests with rabbit anti-B. burgdorferi conjugate.Kidneys, spleens, and samples of blood were examined from 42 live-trapped white-footed mice (Peromyscus leucopus) for B. burgdorferi. No spirochetes were isolated from these organ cultures. Occasional human cases of Lyme disease in Indiana and the observation of spirochetes similar to B. burgdorferi in size and morphology from D. Variabilis ticks, suggest that B. burgdorferi may be present in Indiana.