Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFNγ receptor α chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFNγ receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimere rather than reassociating with the receptor binding site. Our results demonstrate that IFNγ-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.