Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross-reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans.