A Ca(2+)-dependent stimulation of mitochondrial phospholipase A2 is often assumed to play a role in mitochondrial Ca2+ release. We sought to clarify this relation by measuring Ca2+ transport and determining phospholipase A2 reaction products from the same sample of isolated, incubated rat liver mitochondria. When mitochondria had accumulated and spontaneously released again Ca2+, most probably by membrane permeability transition, there was no increase of phospholipase A2 reaction products. However, when the incubation was continued after Ca2+ release, significant increases of the content of lysophosphatidylcholine and unesterified fatty acids could be seen. Quinacrine, an inhibitor of phospholipase A2 activity, prevented Ca2+ release and p-hydroxymercuribenzoic acid, an inhibitor of lysophospholipid reesterification, induced a fast release of Ca2+ from isolated mitochondria. Such effects are usually taken as indirect evidence for a participation of phospholipase A2 in mitochondrial Ca2+ release, but analysis of the mitochondrial lipids revealed that no significant changes of the mass of phospholipase A2 reaction products had occurred. These experiments suggest that the accumulation of phospholipase A2 reaction products in mitochondria is the consequence rather than the cause of the membrane permeability transition. Exogenous phospholipase A2 products, lysophosphatidylcholine and arachidonic acid, induced mitochondrial Ca2+ release after a time lag, which decreased with aging of the mitochondrial preparation. The amount of lysophosphatidylcholine taken up by the mitochondria from the incubation medium during these experiments was measured and compared to the amount of lysophosphatidylcholine produced endogenously by mitochondrial phospholipase A2. From these data it appears likely that the amount of lysophosphatidylcholine generated in the mitochondria after the permeability transition is sufficient to sustain the permeable state. An accumulation of mitochondrially generated phospholipase A2 reaction products after the permeability transition could thus be a decisive factor for the limited reversibility of the membrane permeability transition.