The limited accessibility of specimens of Onchocerca volvulus has hampered investigations of factors that determine the clinical course of infection of man by this filarial nematode. Parasites antigenically related to O. volvulus have been variously proposed as alternative sources of test antigens. The present study has utilized serologic methods to probe the degree of identity of the antigenic constituents of O. volvulus and Dirofilaria immitis, the dog heartworm. 35S-methionine-labeled proteins of dog heartworm microfilariae (mf) were analyzed in immunoprecipitation assays by using a panel of sera drawn from humans diagnosed for onchocerciasis and from canines infected with D. immitis. Unique and common antigens detected by these sera were resolved in polyacrylamide gels. The majority of high-titered human and infected dog sera reacted with antigenic molecules estimated at 15, 16, 28, 42, 54, 66, and 100 kilodaltons. In addition, a subset of these sera detected large amounts of a 75 kilodalton antigen. Human anti-O. volvulus antibodies were found to bind to intact glutaraldehyde-fixed mf of D. immitis in immunoperoxidase assays. In enzyme-linked immunosorbent assays, the same sera reacted with extracts of three distinct developmental stages of the canine pathogen--namely, the mf, embryonated eggs, and adult worms. Preincubation of dirofilarial antigens with human anti-O. volvulus sera inhibited the binding of dog anti-heartworm serum by as much as 70%. Sera of guinea pigs hyperimmunized with mf of O. volvulus inhibited the binding of the immune dog serum to the same extent. These results represent evidence for extensive homology in the antigenic composition of O. volvulus and D. immitis.