Abstract Monoclonal antibodies have been raised against human heart- and brain-type creatine kinase (CK-MB and CK-BB). We used a low-affinity monoclonal antibody to develop a simple two-step immunoaffinity purification procedure for native CK-MB. Antibodies of higher affinity were used to construct specific two-site immunoradiometric assays for CK-MB and CK-BB. In the assay for CK-MB we used an 125I-labeled B subunit-specific antibody with an immobilized anti-M subunit antibody--adding either simultaneously, for a 1-h assay in which between 5 and 1000 ng of CK-MB per milliliter could be measured with an intra-assay CV of 4% to 20%, or sequentially, for a 2-h magnetic separation assay in which between 0.5 and 1000 ng/mL could be measured with an intra-assay CV of 14 to 19%. In both versions CK-BB up to 100 ng/mL and CK-MM up to 5000 ng/mL did not interfere. In the assay for CK-BB we used an 125I-labeled, reduced, and alkylated monoclonal antibody specific for the B subunit of CK-BB, and removed bound isoenzyme with a second immobilized monoclonal antibody specific for a different epitope on the B subunit. Total incubation time for this assay was 5 h. Intra-assay CV was 7.5 to 20% between 0.1 and 1000 ng/mL. CK-MB up to 1000 ng/mL and CK-MM up to 100 000 ng/mL did not interfere. Inter-assay CVs in all three assays varied from 9 to 21%.