Effects of glucagon on cytoplasmic concentration of free calcium, [Ca2+]c, were studied in aequorin-loaded hepatocytes. Addition of 5 nmol/l glucagon resulted in a prompt, but transient increase in aequorin bioluminescence. Glucagon, at 5 nmol/l, induced an increase in [Ca2+]c even in medium containing 1 mumol/l calcium, although the response was considerably smaller than that observed in medium containing 1.0 mmol/l calcium. When hepatocytes incubated in the presence of 1 mumol/l extracellular calcium were first stimulated by phenylephrine and subsequently by either glucagon or angiotensin II, there was a response of [Ca2+]c to glucagon, but not to angiotensin II. Dantrolene (50 mumol/l), which inhibits an increase in [Ca2+]c induced by phenylephrine, did not inhibit the increase in [Ca2+]c induced by glucagon. In contrast, dinitrophenol (50 mumol/l) abolished [Ca2+]c response to glucagon without abolishing the increase in [Ca2+]c induced by angiotensin II. These results suggest that glucagon mobilizes calcium from both intracellular and extracellular pools and that the intracellular calcium pool involved in glucagon action may be different from that mobilized by either phenylephrine or angiotensin II.