Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.