The metabolic action of glucagon in the different spaces that can be reached via the hepatic artery in the bivascularly perfused rat liver of fed rats was investigated. When perfusion was performed in the anterograde mode, glucagon (10 mM) was infused either into the portal vein (type 1 experiment) or into the hepatic artery (type 2); in the retrograde mode, the hormone was infused either into the hepatic vein (type 3) or into the hepatic artery (type 4). The aqueous cell spaces were measured by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis (lactate plus pyruvate production) were measured as metabolic parameters. The following results were obtained. (1) The aqueous cell space accessible via the hepatic artery in the type 2 experiment was 0.63 ml/g; in the type 4 experiment this space was 0.18 ml/g (only periportal cells); glucagon up to 10 nM did not affect these cellular spaces nor did it affect the vascular spaces. (2) The effects of glucagon on glucose release, oxygen uptake and glycolysis were practically the same in all types of experiment (1 to 4), i.e., the action of glucagon was not a function of the accessible cell spaces. (3) When the respiratory chain of the liver cells accessible via the hepatic artery in the type 4 experiment was inhibited by cyanide, glucagon still increased oxygen uptake; oxygen uptake stimulation by glucagon was completely blocked only when cyanide was given to all liver cells. (4) Calcium depletion did not affect the action of glucagon on glucose release and oxygen uptake in the type 4 experiment. It was concluded that, in addition to the receptor-elicited response, the action of glucagon can also be propagated by cell-to-cell communication.