Diglycerides (DGs) are phospholipid-derived second messengers that regulate PKC-dependent signaling pathways. Distinct species of diglycerides are generated from inflammatory cytokines and growth factors. Growth factors increase diacyl-, but not ether-linked DG species, whereas inflammatory cytokines predominately generate alkyl, acyl- and alkenyl, acyl-linked DG species in rat mesenchymal cells. These DG species have been shown to differentially regulate protein kinase C (PKC) isotypes. Ester-linked diacylglycerols activate PKC and cellular proliferation, in contrast to ether-linked DGs, which lead to growth arrest through inactivation of PKC. It is now hypothesized that ether-linked DGs inhibit mitogenesis through inactivation of ERK and/or AKT signaling cascades. We demonstrate that cell-permeable ether-linked DGs reduce vascular smooth muscle cell growth by inhibiting platelet-derived growth factor-stimulated ERK in a PKC- dependent manner. This inhibition is specific to the ERK pathway, as ether-linked DGs do not affect growth factor-induced activation of other family members of the MAP kinases, including p38 MAPK and c-Jun N-terminal kinases. We also demonstrate that ether-linked DG reduce pro-survival PI3K/AKT signaling, independent of PKC, by diminishing an interaction between the subunits of phosphatidylinositol-3-kinase (PI₃K) and not by affecting protein (PP2A) or lipid (PTEN) phosphatases. Taken together, our studies identify ether-linked DGs as potential adjuvant therapies to limit vascular smooth muscle migration and mitogenesis in atherosclerotic and restenotic models.