Mitochondria contribute to cytosolic Ca(2+) homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAG's) induce Ca(2+) release from Ca(2+)-loaded mammalian mitochondria. Release is not mediated by the uni-porter or the Na(+)/Ca(2+) exchanger, nor is it attributed to putative catabolites. DAG's-induced Ca(2+) efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca(2+) and relatively slow reuptake, a secondary progressive release of Ca(2+) occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAG's-induced Ca(2+) efflux is abolished by La(3+) (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca(2+) efflux. Ca(2+)-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAG's-induced Ca(2+) release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAG's-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La(3+). The presence of a second messenger-sensitive Ca(2+) release mechanism in mitochondria could have an important impact on intracellular Ca(2+) homeostasis.