The gene for the second chain of the human interferon-gamma receptor was analyzed from cosmid DNA clones. The gene spans over 33 kilobases of DNA and contains seven exons. The signal peptide is encoded by exons 1 and 2, the extracellular domain by exons 2, 3, 4, 5, and by part of 6. Exon 6 also encodes the whole transmembrane domain and part of the intracellular domain. Exon 7 encodes the remainder of the intracellular domain and contains the 3'-untranslated region. The sequences at the exon/intron boundaries are well conserved with respect to canonical acceptor/donor sites (AG/GT). The 5'-flanking region was sequenced and analyzed for transcription factor binding sites. No TATA or CAAT boxes in the promoter region were identified. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites. Promoter activity of the 5'-flanking region was investigated with a luciferase reporter gene and the cytomegalovirus minimal promoter. Segments of the 5' region with promoter activity were identified.