The glycine cleavage enzyme system (GCS) is found in mitochondria. In liver it is activated by glucagon and other hormones but it is not known how the hormonal signal is transmitted to the mitochondria. We found that the cell-permeant protein phosphatase inhibitor okadaic acid stimulated flux through GCS and could induce a significant increase in the sensitivity of GCS and of glycogenolysis to glucagon. Half-maximal stimulation of GCS by glucagon occurred at 3.2±0.6 nM, whereas it was fully activated at 0.3 nM in the presence of 1 μM okadaic acid. The protein kinase A agonist adenosine-3ʹ,5ʹ-cyclic monophosphorothioate, Sp isomer (10 μM) stimulated the GCS flux by approx. 100%. This stimulation was inhibited by the protein kinase A antagonist 8-bromoadenosine-3ʹ,5ʹ-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS). Although Rp-8-Br-cAMPS significantly inhibited glucagon-stimulated glycogenolysis it had no effect on the glucagon-stimulated GCS flux. These results indicate that a cytoplasmic phosphorylated protein is involved in transmitting glucagon's effect to the mitochondria. However, protein kinase A does not have a necessary role in transmitting glucagon's signal. We also examined the role of protein kinase C because angiotensin II also stimulated flux through GCS. However, the phorbol ester PMA had no effect on either GCS or on glycogenolysis.