Phosphorylation of endogeneous phosholipids of rat liver mitochondrial fractions with γ[32P]ATP revealed formation of all the known inositol phospholipids, such as phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. Additionally, a new inositol phospholipid was detected. Incorporation of [3H]-labelled insositol followed a similar profile. Enzymatic experiments indicated that the new lipid could possibly be phosphatidylinositol trisphosphate. The presence of phosphoinositides-generated second messengers such as diacylglycerol and inositol trisphosphate was also confirmed. Protein kinase C, which acts as mediator between second messengers and nuclear factors, was also found to be present in mitochondria in significant amount. These results suggest that phosphoinositide signal transduction pathway is operative in rat liver mitochondria.