The anterior organelles of the coccidian parasite Toxoplasma gondii have long been suspected of playing a role in the ability of this organism to actively penetrate a wide range of host cells. A series of four monoclonal antibodies (produced by spleen cells from mice immunized with whole, killed T. gondii fused with Sp 2/0-Ag14 myeloma cells) recognized anterior organelles of T. gondii in indirect immunofluorescence assays. These antibodies (Tg 13, Tg 31, Tg 49, and Tg 112) were of the immunoglobulin G (IgG) class, had different enzyme-linked immunosorbent assay titers, and partially competed with each other in a solid-phase immunoassay with whole, dried T. gondii as the antigen. It was observed by immunofluorescence that all antibodies detected anterior structures, which under some conditions of fixation and extraction appeared to be multiple rodlike organelles resembling rhoptries. As determined by ultrastructure immunocytology, Tg 49 recognized electron-dense bodies consistent with rhoptries or micronemes in parasites that had been fixed in 2% paraformaldehyde and extracted with Triton X-100 to allow antibody penetration. An assay of penetration enhancement, in which conditioned medium (from fibroblast monolayers completely lysed by T. gondii) increased the number of plaques produced by a standard inoculum of T. gondii on fresh monolayers, was inhibited by equal amounts of all four monoclonal antibodies, in degrees closely related to their enzyme-linked immunosorbent assay titers. These antibodies appeared to link a penetration-enhancing factor with the rhoptries of T. gondii.