The expression pattern of diacylglycerol kinase (DGK) and the biological significance of DGKepsilon in vascular smooth muscle cells were investigated. mRNA expression for DGKalpha, DGKepsilon, and DGKzeta was detected in isolated rat aortic smooth muscle cells (RASMCs) and A7r5 cells by reverse transcription with polymerase chain reaction analysis. An immunocytochemical study revealed intense DGKepsilon in a filamentous pattern, parallel to the long axis of cell, and on actin stress fibers as shown by double-staining with fluorescent phalloidin. DGKalpha was detected sparsely in the cytoplasm and nucleus, and DGKzeta was observed as a granular pattern in the nucleus. In order to elucidate the functional significance of DGKepsilon, its immunoreactivity was examined in RASMCs incubated with serotonin, a vasoconstrictive agonist. When RASMCs were stimulated with serotonin, the cells lost their polarization and shortened, i.e., contracted. In RASMCs contracted by serotonin, DGKepsilon was detected diffusely in the cytoplasm without a filamentous stress fiber pattern. Protein and mRNA expression of DGKepsilon in RASMCs was significantly increased by stimulation with serotonin. Inhibition of Rho-associated kinases by Y-27632 or inhibition of actin polymerization by cytochalasin B resulted in a decrease in the intensity of DGKepsilon immunoreactivity on stress fibers. The results suggest that DGKepsilon interacts with actin stress fibers and is involved in their stability in vascular smooth muscle cells.