Recent evidence suggests that reactive oxygen species (ROS) may function as second messengers in intracellular signal transduction pathways. We explored the possibility that ROS were involved in lysophosphatidic acid (LPA)-induced mitogen-activated protein (MAP) kinase signaling pathway in HeLa cells. Antioxidant N-acetylcysteine inhibited the LPA-stimulated MAP kinase kinase activity. Direct exposure of HeLa cells to hydrogen peroxide resulted in a concentration- and time-dependent activation of MAP kinase kinase. Inhibition of catalase with aminotriazole enhanced the effect of LPA on induction of MAP kinase kinase. Further, LPA stimulated ROS production in HeLa cells. These findings suggest that ROS participate in the LPA-elicited MAP kinase signaling pathway.