In this report, we have demonstrated that IFN-gamma and TNF-alpha increase expression of both the I-A and I-E region gene products on the surface of the myelomonocytic cell line WEHI-3, and that they mediate this increase via an increase in A alpha transcription. Constructs containing 5' deletion mutations of the A alpha promoter attached to the bacterial chloramphenicol acetyl transferase gene were used to delineate the minimum 5' flanking sequences required for promoter activity, and for inducibility by IFN-gamma and TNF-alpha. Approximately 115 bp of 5' sequences are required for minimum induction by IFN-gamma or TNF-alpha when the cytokines are present separately. This includes the three conserved promoter elements, the X, Y, and H boxes. Nested linker-scanner mutations demonstrated that additional regions were also critical for optimal induction by IFN-gamma or TNF-alpha. These include the kappa B-like enhancer and a TNF-alpha-specific sequence that we have tentatively called the T box. The T box sequence was also found in the promoter regions of the human HLA-DQ alpha and rat RT1.B alpha genes. Although the entire T box sequence element was not found in the other mouse class II genes, all class II alpha genes contained the SV40 core enhancer element in the regions included by the T box. Mouse class II beta genes appear to contain neither the T box nor the core enhancer element in this region, suggesting differential regulation of class II alpha and beta genes by TNF-alpha.