Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Phoneutria nigriventer spider venom. Horse anti-P. nigriventer immunoglobulins were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with P. nigriventer venom from those in mice inoculated with Lycosa sp. and Loxosceles intermedia spider venoms, Tityus serrulatus scorpion venom and Apis mellifera bee venom. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice and to detect antigens in the sera of patients envenomed by P. nigriventer. © 2001 Elsevier Science Ltd.