Isolated rat hepatocytes do not actively accumulate Ca2+ during prolonged incubation in vitro; however, these cells do exhibit a limited exchange of intracellular with extracellular Ca2+. The exchangeable pool represents about 2 nmol of CaS+ per mg of protein. In medium containing either a low (20 MM) or high (1 mM) concentration of Ca2+, the divalent cation ionophore, A23187 (at concentrations of 0.03-0.1 nmol/ mg of protein), causes release of 42Cad+ from this exchangeable pool but does not allow net influx of extracellular CaZ+ detectable by the use of a Ca2+-sensitive electrode. Like A23187, the hormones norepinephrine, vasopressin, and glucagon (at concentrations that stimulate gluconeogenesis) each induces a similar net efflux of Ca2+. Treatment with one hormone decreases the subsequent reponse to the others, whereas treatment with A23187 abolishes the hormonal effects upon both Ca2. release and gluconeogenesis. The action of norepinephrine, but not of glucagon, upon CaO+ efflux is prevented by the a-adrenergic antagonist, phenoxybenzamine. The action of norepinephrine is not prevented by the ,B-adrenergic antagonist, propranolol Together these results indicate that the release of Ca2+ from a common pool of exchangeable Ca2+ is important to the action of a variety of hormones on hepatocytes. This Ca2+ pool in the isolated hepatocyte is characterized as being similar in size and having exchange kinetics that are com arable to those reported for the major intracellular pool of Ca + in the intact liver. The possibility that this pool is intramitochondrial calcium is discussed.The effects of glucagon upon gluconeogenesis in intact, perfused liver are mediated through alterations in cyclic nucleotide levels (1,2 Zahlten and Stratman (11). Routinely, cells were suspended at 6.5 X 106 cells per ml in Krebs-Henseleit bicarbonate medium containing 1.5% bovine serum albumin, 10 mM Na lactate, and 1 mM Na pyruvate without added calcium (the free calcium in the cell suspension was estimated at 15-30 gM). Cell suspensions (1.0 or 2.5 ml) were incubated in plastic scintillation vials at 370 in a Dubnoff shaker with agitation (110 cycle/min) under 95% 02/5% CO2.45Ca2+ Exchange Studies. After 40 min of equilibration, 10 gCi of 45Ca2+ was added to each 1.0 ml of cell suspension. Subsequently 20-,ul aliquots were removed for determination of isotopic Ca uptake. Each aliquot was layered on the top of 0.35 ml of 7% bovine serum albumin in 5 mM CaCl2/150 mM NaCl all contained in a 0.45-ml polyethylene centrifuge tube (no. 47/7, Sarstedt Co., Princeton, NJ). The tubes were then centrifuged for 15 sec at 2000 X g. The tips containing the cell pellet were cut off and their contents were flushed into scintillation vials by injecting 0.5 ml of 1% sodium dodecyl sulfate solution. The cells were solubilized with 5 ml of Aquasol and the associated radioactivity was determined.Ca2+ Electrode Studies. Cell suspensions of 2.5 ml were prepared as described above with each vial containing a Ca2+-sensitive and a reference electrod...