The HLA-A, -B, and -C class I human histocompatibility antigens and the genes that encode them have been isolated and characterized. Apparently complete class I non-HLA-A,B,C genes have been identified on HindU-generated 5.4-kilobase (kb), 6.0-kb, and 6.2-kb DNA fragments derived from lymphoblastoid cell line (LCL) 721. We studied the expressibility of these genes by subcloning them into the nonintegrating pHeBo vector and transferring the chimeric plasmids into mutant LCL 721. lysates showed that transfer of each non-HL4-AB,C class I gene into 721.221 resulted in the appearance of an a chain that coprecipitated with P2-microglobulin. The three previously unreported a chains differed from each other in size and were smaller than HLA-A, -B, and -C a chains. These observations clearly show that these three cloned, nonallelic, non-HLA-A,B,C class I genes encode a chains that can be expressed in human cells.