Lyme disease recently has been recognized in Wisconsin. Trapping studies were conducted at four geographically separate and ecologically distinct regions in Wisconsin to elucidate the distribution and host preferences of Ixodes dammini on small and medium sized mammals, and the occurrence of antibodies to Borrelia burgdorferi in these wild mammals. Peak I. dammini larval activity occurred from June–September. Nymphs were most active from May–August. White-footed mice (Peromyscus leucopus) and chipmunks (Tamias striatus) were important hosts for immature ticks. Mean numbers of I. dammini per mouse were highest in regions of high prevalence of Lyme disease. Antibody to B. burgdorferi was detected in sera of 60/371 (16%) white-footed mice, 5/104 (5%) chipmunks, 3/5 (60%) gray squirrels (Sciurus carolinensis), 0/8 raccoons (Procyon lotor), and 0/12 opossum (Didelphis virginiana); antibody prevalence correlated positively with I. dammini occurrence, and seropositive animals were not detected in areas where I. dammini were not found. Two of 15 recaptured P. leucopus had ≥4-fold changes in antibody titer. B. burgdorferi was cultured from blood of a P. leucopus captured in west-central Wisconsin, and was observed by direct immunofluorescence in 9/23 (39%) I. dammini nymphs. In Wisconsin, I. dammini has increased in numbers and has significantly expanded its range since its first recognition in 1968.