Binding of glucagon and glucagon-stimulated cyclic AMP production were studied in highly purified liver plasma membranes from growing rats fed a 12% protein diet (group 1) or 20% protein diet; this latter was given either in normal (group 2) or restricted (group 3) amounts. Groups 2 and 3 exhibited significantly higher peripheral glucagonemia than group 1 (amounting to 286 and 160% of group 1, respectively). Specific [125I]iodoglucagon binding to plasma membranes was similar in all groups. Scatchard analysis revealed no further differences between affinity constants and binding capacities in the three groups. Hormone degradation was constant. As membrane recovery and membrane purity were unaltered, these results suggest that hyperglucagonemia caused by increasing dietary protein level is not associated with any significant modification of glucagon binding sites in rat liver. In the presence of a potent phosphodiesterase inhibitor (3-isobutyl-1-methyl xanthine 0.2 mM) the glucagon-stimulated cyclic AMP production was higher in rats fed the 20% protein diet, which was given in normal amounts, than in rats fed the 12% protein diet. In contrast when the 20% protein diet was given in restricted amounts the glucagon-stimulated cyclic AMP production was similar to that in the 12% protein-fed rats.