Rat liver and heart membranes were tested for adenylate cyclase activation by glucagon and 10 glucagon analogs mono- or polysubstituted in positions 2-4, 25, 27 and/or 29. The first membranes were, in addition, examined for the capacity of glucagon analogs to inhibit the binding of [125I]iodoglucagon. The monophasic slope of dose-effect curves suggested interaction with one class of glucagon receptors in both tissues, receptors in liver being more sensitive to the ligands and more efficiently coupled to adenylate cyclase than heart receptors. Structure-activity studies on liver membranes revealed that modifications of the beta-turn potential in the 2-4 region by single residue substitutions could lead to partial agonists (with D-Gln3 or Phe4) or to a superagonist (with D-Phe4). The importance of a proper alpha-helix conformation in the C-terminal part of glucagon for binding affinity was also obvious: replacing Trp25, Met27 and Thr29 in combination by Phe25, Leu27 and Thr29-NH2 increased the affinity while single or combined substitutions with Gly25 and/or Nle27 sharply decreased the affinity. Similar trends were less evident but still obvious on heart membranes.