Borrelia burgdorferi dissemination to selected target organs was examined on days 1, 2, 3, 4, 7, 10, 15, 21, and 30 after intradermal inoculation of 4-week-old C3H mice. Infection was determined by culture (blood, spleen, kidney, ear punch); polymerase chain reaction (PCR) for outer surface protein A (OSP A) DNA (ear punch); histology and spirochete histochemistry (spleen, kidney, skin, heart, joints); and OSP A DNA in situ hybridization (joints, heart). Blood or spleen of most mice were culture positive by day 3 and ear punch by day 10. Polymerase chain reaction performed on ear punches was also positive by day 10. Inflammation of joints and tendons began on days 4 through 7 and heart on days 7 through 10, which coincided with colonization of tissues with spirochetes. Spirochetes were multifocal in distribution, with a predilection for collagenous connective tissue of joints, heart, arteries, nerves, muscle, skin, and other tissues. Relative numbers of spirochetes peaked at 15 days, then decreased by 21 days. Gamma M immunoglobulin (IgM) antibody was detectable on immunoblots as early as day 4, with subsequent declining reactivity, and IgG antibody was detectable by day 7, with expanding reactivity to multiple antigens through day 30.