Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.