To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 °C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p > GTP > GDP ≫ GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 n m for glucagon and receptor in the absence of GTP, 18.6 n m for glucagon and receptor in the presence of GTP, 1.55 μ m for the association of receptor and GTP presumably linked to an N protein, and 8.86 μ m for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein. Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the β-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.