An enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis, in deer (Odocoileus virginianus) and raccoons (Procyon lotor). Blood samples were collected from these mammals in Connecticut, Maryland, North Carolina, Georgia and Florida. Seropositivity for deer was highest in Connecticut (56% of 353 sera) and Maryland (51% of 35 sera). Raccoons in Connecticut, Maryland, North Carolina, and Florida also had antibodies to B. burgdorferi, but prevalence of positive sera was highest in Maryland (79% of 14 samples). Based on adsorption tests, the immunoglobulins detected in these mammals were probably specific to B. burgdorferi. The ELISA was more sensitive than an indirect fluorescent antibody staining method and was more suitable for analyzing large numbers of serum samples.