Regulation of HLA class I and class II antigen expression was studied in hybrids of human T and B lymphoblastoid cell lines (LCL). The T-LCL CEM^R.3 expresses no HLA class II antigens. It expresses little total HLA class I antigen and no HLA-B antigens. The B-LCL 721.174 is a radiation-induced variant immunoselected for loss of class II antigen expression. In addition to showing a deletion of all HLA-DR and DQ structural genes, 721.174 expresses no HLA-B antigens and a decreased level of HLA-A antigen compared with the parental cell line. A hybrid of 721.174 and CEM^R.3 expresses class II antigens encoded by CEM^R.3. Increased expression of HLA class I antigens encoded by both 721.174 and CEM^R.3 was also observed. Specifically, the previously undetectable HLA-B5 and HLA-Bw6 antigens encoded by 721.174 and CEM^R.3, respectively, were present on the hybrid. Increased expression of the HLA-A2 antigen encoded by 721.174 was also observed. An immunoselected variant of the hybrid lacking both CEM^R.3-derived copies of chromosome 6 lost expression of the HLA-B5 antigen encoded by 721.174 and expressed a decreased amount of HLA-A2. From these data, we infer that two complementary trans-acting factors mediate enhanced expression of HLA class I antigens in the hybrid. One of these factors is provided by a gene located on chromosome 6, derived from CEM^R.3. The second factor, introduced by 721.174, is the gene previously postulated to induce expression of CEM^R.3-encoded class I antigens in hybrids of CEM^R.3 with B-LCL.