We have shown previously that a four-amino acid block residing at positions 266-269 (LPKS) in the intracellular domain of the human interferon- γ (IFN-γ) receptor α chain is critical for IFN-γ-dependent tyrosine kinase activation and biologic response induction. Herein we show that this sequence is required for the constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia expression system, a receptor α chain-specific monoclonal antibody coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN-γ receptor α chain, (b) a receptor α chain truncation mutant containing only the first 59 intracellular domain amino acids, or (c) a receptor mutant containing alanine substitutions for the functionally irrelevant residues 272-275. In contrast, JAK-1 was not coprecipitated when coexpressed with a receptor α chain mutant containing alanine substitutions for the functionally critical residues 266-269 (LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the only residue obligatorily required for receptor function. In addition, Pro-267 is required for JAK-1 binding. These results thus identify a site in the IFN-γ receptor α chain required for constitutive JAK-1 association and establish that this association is critical for IFN-γ signal transduction.