The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferonγ (IFNγ) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFNγ-receptor α-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT1 is present in a higher molecular weight complex(es) and, at least for IFNγ-primed cells, is available for recruitment to the activated LFN γ-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.