The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca 2؉ accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.Babesia bovis is a hemoprotozoan parasite that causes great economic losses to the cattle industry worldwide. It is transmitted by tick vectors and has an asexual intraerythrocytic cycle in the infected cattle (5, 9). Understanding the basic molecular mechanism(s) of the asexual intraerythrocytic cycle, particularly the process of merozoite invasion into and escape from infected erythrocytes (RBC), may accelerate the development of an effective vaccine. Extracellular merozoites are directly exposed to the host humoral immune components. Consequently, efforts to identify potential components for the development of a vaccine are primarily directed at the merozoite stage.Apicomplexans utilize several rhoptry proteins in their invasion into and development within the host cell (16, 18). Extracellular merozoites attach to the host RBC and reorient to bring the apical organelles close to the attachment interface, and through the interaction of protozoan ligands with several surface receptors, the rhoptry products are released at the point of membrane invagination. Although the morphological events during host cell recognition and penetration appear to be similar among the apicomplexans, some molecular events mediated by each secretory component of the rhoptry vary and are unique (16,18).The rhoptry-associated protein 1 (RAP-1) of B. bovis merozoites bears substantial sequence homology to the RAP-1 of other Babesia parasites (3, 4) and contains immunogenic B-cell epitopes (20), and the purified recombinant RAP-1 has proven effective in inducing protective immunity in the vaccinated cattle (22). All of these earlier findings point to the biological and immunological characteristics of RAP-1 that would justify its inc...