The present study was undertaken to test the hypothesis that a high-fat diet-induced liver lipid infiltration is associated with a reduction of hepatic glucagon receptor density (Bmax) and affinity (Kd) and with a decrease in stimulatory G-protein (Gs) content while enhancing inhibitory G-protein (Gi₂) expression. We also hypothesized that, under this dietary condition, a single bout of endurance exercise would restore hepatic glucagon receptor parameters and G protein expression to standard levels. Female Sprague-Dawley rats were either fed a standard (SD) or a high-fat diet (HF; 40% kcal) for two weeks (n = 20 rats/group). Each dietary group was, thereafter, subdivided into a non-exercised (Rest) and an acute exercised group (Ac-Ex). The acute exercise consisted of a single bout of endurance exercise on a treadmill (30 min, 26 m/min, 0% slope) immediately before sacrifice. The HF compared to the SD diet was associated with significantly (P < 0.05) higher values in hepatic triglyceride concentrations (123%), fat pad weight, and plasma FFA concentrations. The high-fat diet also resulted in significantly (P < 0.05) lower hepatic glucagon receptor density (45 %) and Gs protein content (75%) as well as higher (P < 0.05) Gi₂ protein content (27%) with no significant effects on glucagon receptor affinity. Comparisons of all individual liver triglyceride and Bmax values revealed that liver triglycerides were highly (P < 0.003) predictive of the decreased glucagon receptor density (R = - 0.512). Although the 30-min exercise bout resulted in some typical exercise effects (P < 0.05) such as an increase in FFA (SD diet), a decrease in insulin levels, and an increase in plasma glucagon concentrations (SD diet), it did not change any of the responses related to liver glucagon receptors and G proteins with the exception of a significant (P < 0.05) decrease in Gi₂ protein content under the HF diet. The present results indicate that the feeding of a high-fat diet is associated with a reduction in plasma membrane hepatic glucagon receptor density and Gs protein content, which is not attenuated by a 30-min exercise bout. It is suggested that liver lipid infiltration plays a role in reducing glucagon action in the liver through a reduction in glucagon receptor density and glucagon-mediated signal transduction.