HLA Class II-negative severe combined immunodeficiency (SCID) results from a congenital defect characterized by an absence of HLA Class II antigens. Patients with the disorder have no HLA-DR, DQ, or DP antigens or mRNAs in their peripheral-blood lymphocytes. The affected gene is a recessive, transacting regulatory gene that controls the expression of Class II genes. We studied the regulation of HLA Class II gene expression with the use of established Epstein-Barr virus-transformed B-cell lines and skin fibroblast lines from a group of patients with SCID. Lymphoblastoid B-cell lines from the patients contained no mRNA for HLA-DR, DQ, and DP alpha and beta polypeptides, but did express mRNA for the HLA-associated invariant chain, which is normally coregulated with HLA Class II antigens. In the B-cell line from one patient, a very low amount of DR mRNA could be detected, indicating some heterogeneity in SCID. The lymphokine gamma-interferon, a strong inducer of Class II genes in a variety of normal cells, did not restore Class II gene expression in any of the SCID B-cell lines. More important, gamma-interferon was unable to induce any Class II mRNA in fibroblast lines from patients with SCID, in contrast to the efficient induction observed in normal fibroblasts. The invariant-chain gene, however, was induced in the SCID fibroblasts, confirming a unique uncoupling in the regulation of invariant and Class II genes. Thus, the genetic defect in patients with SCID affects not only the B-cell lineage but also the inducible expression of HLA Class II genes that is normally observed in Class II-negative cells, such as fibroblasts. This unresponsiveness to gamma-interferon in vitro indicates that patients with SCID will not respond to treatment with this lymphokine. Our data also increase understanding of the normal mechanisms regulating the genes for the HLA Class II cell-surface glycoproteins.