Several factors for the hydrogen peroxide (H2O2)-induced PLD stimulation have been proposed, including protein kinase C (PKC), tyrosine kinase, mitogen-activated protein kinase and Ca2+, but their precise roles remain to be defined. As for involvement of PKC, there has been some discrepancy. Our previous study has demonstrated that phospholipase D (PLD) activity was increased by exposure of PC12 cells to 0.5mM H2O2 in modified Krebs-Ringer buffer (KRB) and suggested that the PLD activation was independent of PKC activity. However, we have shown here that the H2O2-induced PLD stimulation was much greatly enhanced by incubation in Dulbecco's modified Eagle's medium (DMEM) and further that it was PKC-dependent. These results indicated that the markedly enhanced PLD activation and its PKC dependence were modulated by pH changes during incubation in DMEM. Furthermore, evidence has been presented for possible involvement of alkaline phosphatase in this pH-dependent profile of PLD activation by H2O2. (C) 2003 Elsevier Inc. All rights reserved.