Vitamin K3 (menadione), a synthetic vitamin K congener, inhibits the growth of tumor cells. Here, we examined possible effects of vitamin K3 on phospholipase D (PLD) activity, an enzyme which produces growth regulatory substances. In NIH 3T3 fibroblasts, vitamin K3 (50-100 microM) alone had no effect on PLD-catalyzed formation of phosphatidylethanol, a marker of PLD activity, but it slightly (10-21%) inhibited the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Of the two major substrates of PLD, phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), vitamin K3 (10-100 microM) preferentially inhibited PtdEtn hydrolysis when stimulated by PMA or platelet-derived growth factor, the latter being a hormonal activator of PKC. Vitamin K3 had no inhibitory effect on sphingosine- or staurosporine-induced hydrolysis of PtdEtn or PtdCho. Inhibition of PMA-induced PtdEtn hydrolysis by vitamin K3 was effectively reduced by both cysteine (1 mM) and reduced glutathione (1 mM) and was mimicked by the superoxide-generating xanthine/xanthine oxidase system. The results show that vitamin K3 preferentially inhibits the effects of PKC activators on PLD-mediated hydrolysis of PtdEtn by a mechanism which may involve oxidation of thiols in a critically important regulatory component.