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 μM 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 < 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 ± 0.07nmol/mg protein in controls and 0.22 ± 0.03 nmol/mg protein (P < 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 of mitochondria. These results suggest that α-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 α-adrenergic stimulation.