The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase that plays an important role in the antiviral activities of interferon (IFN). To determine the organization and regulation of the PKR locus, λ phage and bacterial artificial chromosome (BAC) clones containing the human PKR gene were isolated. Characterization of these clones revealed that PKR has 17 exons and 16 introns dispersed in a genomic region of 50 kb. Sequence analysis of the PKR 5'-flanking region identified a canonical IFN-stimulated response element (ISRE), GAAAACGAAACT. Transient transfection of PKR promoter constructs linked to a luciferase reporter gene into human T98G cells indicated that this 5'-flanking region is capable of functioning as a basal promoter that is also inducible by IFN-α and IFN-β but not IFN-γ. Interestingly, the PKR gene contains a polymorphic CGG trinucleotide repeat in exon 1. In addition, four PKR alleles, varying in repeat number from 7 to 10, were detected in 30 individual chromosomes. The PKR gene undergoes alternative splicing of exon 2, which gives rise to two forms of 5'-untranslated exons of different length. Although the human and murine PKR proteins have high homology, comparison of their gene structures reveals divergence in 5'-flanking regions, suggesting distinct regulation at the genomic level.