Glucagon treatment of chick hepatocytes in primary culture results in a rapid 20–30% decrease in NaF-stimulated adenylyl cyclase activity. This heterologous desensitization is rapidly reversible upon removal of hormone from the culture medium. This decrease in NaF-stimulated adenylyl cyclase activity agrees well with the decrease in Gs activity, as assessed by reconstitution of the cyc− S49 cell adenylyl cyclase. Recovery from heterologous desensitization within 30 min results in restoration of Gs activity to control levels. No changes in Gs-α protein levels could be determined by immunoblotting using an αs-specific antiserum during heterologous desensitization. Similarly, no change in the βγ-subunits or Gi-α-subunits were detected by immunoblotting analysis using subunit-specific antisera. Pretreatment of cells with pertussis toxin did not block the onset of either heterologous or homologous desensitization, indicating the lack of involvement of Gi in glucagon-induced desensitization. cAMP-dependent phosphorylation of G-proteins does not account for heterologous desensitization, since none of the G-protein subunits appears to be phosphorylated even in vitro. However, Gs as the locus of change in heterologous desensitization was confirmed by the observation that addition of purified Gs to desensitized cell membranes results in enhancement of NaF-stimulated adenylyl cyclase activity. From these data we conclude that postreceptor heterologous desensitization in chick hepatocytes results from a reversible decrease in Gs activity in the cell surface membrane.