AbstractDecrease in intracellular thiols leads to oxidative stress and thus may cause alterations in the activity of redox‐sensitive enzymes required for signal transduction. Here, we report that, N‐ethylmaleimide and phenylarsine oxide, which are known to oxidize free thiols as well as protein thiols, induced phosphatidyl ethanol generation in the micromolar range suggesting activation of phospholipase D in vascular smooth muscle cells. These agents also induced significant phosphatidic acid and diacylglycerol generation without causing protein kinase C activation. Phenylarsine oxide and N‐ethyl maleimide induced phosphotipase D activation is protein kinase C independent as it was not inhibited by compound‐3 and bisindolylmaleimide, potent protein kinase C inhibitors. Tyrosine kinase inhibitor herbimycin A by itself activated PLD, but inhibited the phospholipase D activation by phenytarsine oxide and N‐ethylmaleimide. These results suggest that oxidation of the cellular thiols activates phospholipase D independent of protein kinase C.