Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.