The incubation of isolated hepatocytes with the inhibitor of protein mono ADP-ribosylation, m-iodobenzylguanidine (MIBG), resulted in an increase in the size of the mitochondrial Ca2+ pool, without alteration of the non-mitochondrial Ca2+ store(s). This increase was abolished when the cytosolic free Ca2+ concentration ([Ca2+]i) was buffered by prior loading of the cells with fluo 3. Elevating [Ca2+]i by releasing the endoplasmic reticular Ca2+ store with 2,5-di-(tert-butyl)-1,4-hydroquinone resulted in a synergistic increase in the magnitude of the mitochondrial Ca2+ pool. A role for protein ADP-ribosylation in the intracellular regulation of mitochondrial Ca2+ homeostasis is suggested.