Insulin receptor associated with the cerebral cortex (CC) has been shown to be involved in brain cognitive functions. Furthermore, deterioration of insulin signaling has been associated with age-related brain degeneration. We have reported previously that aging stimulates phospholipase D/phosphatidate phosphohydrolase 2 (PLD/PAP2) pathway in CC synaptosomes from aged rats, generating a differential availability of their reaction products: diacylglycerol (DAG) and phosphatidic acid (PA). The aim of this work was to determine the effect of aging on DAG kinase (DAGK), as an alternative pathway for PA generation, and to evaluate the effect of insulin on PLD/PAP2 pathway and DAGK. PLD, PAP2, and DAGK activities were measured using specific radiolabeled substrates in CC synaptosomes from adult (4 months old) and aged rats (28 months old). In adult animals, in the presence of the tyrosine phosphatase inhibitor (sodium o-vanadate), insulin stimulated PLD activity at 5 min incubation. DAGK activity was also increased at the same time of incubation and PAP2 was inhibited. In aged animals, PLD activity was not modified by the presence of insulin plus vanadate, PAP2 was inhibited, and DAGK was stimulated by the hormone. Insulin, vanadate, and the combination of both induced protein tyrosine phosphorylation in adult CC synaptosomes. Aged rats showed a lower level of protein phosphorylation with respect to adult rats. Our results show that insulin modulates PA and DAG availability through the regulation of PLD/PAP2 and DAGK pathways in adult rat CC synaptosomes. Additionally, we demonstrated that PA and DAG generation is regulated differentially by insulin during aging.