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A seroepidemiologic study was carried out in humans inhabiting southeastern Puerto Rico, the area on this island with highest prevalence of canine heartworm, to detect the presence of antibodies to Dirofilaria immitis. Three hundred serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) using somatic antigens of the adult worm. Serum samples from eight patients were positive for anti-dirofilarial IgG. These patients had a mean age of 54 years. Although the study population had a female: male of 2.5:1, six out of the eight positive sera were from males. The minimum prevalence of human dirofilariasis was conservatively estimated to be 2.66%. After Western blot analysis of positive sera, four polypeptide markers with Mr values of 15, 33, 42, and 69 kD were selected as potential immunologic markers for this human infection. Three of them (15, 33, and 69 kD) were specifically reactive only with ELISA-positive sera. The 42-kD polypeptide reacted strongly with ELISA-positive sera, although it reacted weakly with some of the ELISA-negative controls. The 15-, 33-, and 69-kD polypeptides were recognized by positive sera in several combinations, but only those sera with the highest ELISA optical density values reacted with all three polypeptides.