We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.