Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.