We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes. Thirty-two previously typed cell lines were used as reference material. The MICA gene frequencies among 201 African-American unrelated donors were determined. Of 51 previously known alleles, 18 were observed in African-Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African-Americans. MICA*00201 and MICA*00801 were the two most frequent alleles in African-Americans. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of human leukocyte antigen-B in the African-American population. The methodology described here offers a powerful new approach to DNA typing of the MICA alleles.