We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.