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A total of 28 double cloned monoclonal antibodies specific for Plasmodium knowlesi were raised by fusion of Y3 rat myeloma cells with spleen cells of A0 rats immunized with W1 variant isolated merozoites. Four of these antibodies reacted positively in a solid phase radioimmunoassay against glutaraldehyde-fixed schizonts but gave no detectable reaction on indirect immunofluorescence against methanol-fixed schizonts or merozoites. The remaining 24 antibodies could be divided into 13 distinctive immunofluorescent categories on the basis of their patterns of binding to schizonts and merozoites and reactivity with Plasmodium falciparum. Eight antibodies were studied for their ability to inhibit the in vitro multiplication of W1 P. knowlesi as assessed by parasite incorporation of (3)H-amino acids and parasite counts. Partially purified antibody preparations from ascitic fluids were all inhibitory for parasite growth; however, when fully purified antibodies were tested, six of the eight proved to be non-inhibitory. Two of the purified antibodies, both IgG2a isotype, inhibited the in vitro multiplication of P. knowlesi in a dose-dependent manner. Inhibition was not associated with detectable damage to intracellular parasites, suggesting that the inhibitory monoclonal antibodies act by blocking the reinfection of red cells by newly released merozoites. On immunofluorescent analysis both inhibitory antibodies bound to methanol-fixed schizonts, with the intensity increasing for progressively more mature parasites; both reacted diffusely with isolated merozoites, and neither cross-reacted with P. falciparum. Both bound specifically to a single metabolically labelled polypeptide which appears to be a minor parasite component and has an approximate molecular weight of 66,000 when analysed by SDS-PAGE fluorography. The putative protective antigen of P. knowlesi has potential interest as a vaccine against P. knowlesi malaria.