A monoclonal antibody (McAb 4A7) raised against a rhoptry enriched subcellular fraction of Toxoplasma gondii tachyzoites reacted in immunofluorescence studies with rod-like organelles located in the anterior part of the organisms and gave specific labeling of rhoptries in immunoelectron microscopy. On immunoblots, two major proteins of 55 and 60 kDa were identified by McAb 4A7. Similar results were obtained both by immunodetection and immunoblotting with tachyzoites, bradyzoites and sporozoites. Pulse chase analysis of [35S]methionine labeled tachyzoites demonstrated that the 55 and 60 kDa rhoptry proteins derived from a 66-68 kDa doublet which was processed approximately 30 min after biosynthesis. Two other monoclonal antibodies (McAb 2F8, McAb 2H3) respectively specific for rhoptry proteins of 55 kDa having a 66 kDa precursor and 60 kDa having a 68 kDa precursor were also obtained; we suggest that they recognize separately the two components of the 55-60 kDa rhoptry protein family of Toxoplasma.