The glycogenolytic effect of catecholamine has been proposed to be mediated by calcium ions mobilized mainly from mitochondria. In this study, the contribution of mitochondria as a source of the mobilized calcium was evaluated by use of perfused rat livers. Phenylephrine-induced efflux of 45Ca from 45Ca preloaded liver was abrupt and transient, and almost ceased within 5 min. The perfusion with 5 microM phenylephrine for 5 min caused a significant decline in the content of 45Ca retained in the liver homogenate; control, 0.97 +/- 0.11 nmol of 45Ca/mg protein (mean +/- SEM) and phenylephrine, 0.41 +/- 0.08 nmol of 45Ca/mg protein (P less than 0.01), when calculated from the specific radioactivity of calcium in the perfusate used for loading perfusion. The contents of 45Ca in mitochondria isolated subsequently from the homogenate were 0.43 +/- 0.07 nmol/mg protein in controls and 0.22 +/- 0.03 nmol/mg protein (p less than 0.05) when phenylephrine was administered. Taking into consideration that mitochondrial protein is a fraction of homogenate protein, it is obvious that the decline in mitochondrial 45Ca represents only a small portion of the total reduction of radioactivity in the homogenate. In a series of experiments in which various perfusion periods were employed for 45Ca loading and for washing-out, it was found that phenylephrine induced 45Ca efflux also from a pool with a calcium turnover rate slower than that if mitochondria. These results suggest that alpha-adrenergic stimulation enhances release of calcium not only from mitochondria but also from other compartments with a slower turnover rate. The probability of plasma membrane is proposed as the other source of calcium released in response to alpha-adrenergic stimulation.