In this paper we demonstrate for the first time a mitogen-induced activation of a nuclear acting phosphatidylcholine-phospholipase D (PLD) which is mediated, at least in part, by the translocation of RhoA to the nucleus. Addition of alpha-thrombin to quiescent IIC9 cells results in an increase in PLD activity in IIC9 nuclei. This is indicated by an increase in the alpha-thrombin-induced production of nuclear phosphatidylethanol in quiescent cells incubated in the presence of ethanol as well as an increase in PLD activity in isolated nuclei. Consistent with our previous report (Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400), the presence of ethanol decreases the alpha-thrombin-induced production of phosphatidic acid without affecting the induced increase in nuclear diglyceride, indicating that the increase in nuclear PLD activity is responsible for the effect on phosphatidic acid, but not that on diglyceride. Our data further demonstrate that RhoA mediates the activation of nuclear PLD. RhoA translocates to the nucleus in response to alpha-thrombin. Additionally, PLD activity in nuclei isolated from alpha-thrombin-treated cells is reduced in a concentration-dependent fashion by incubation with RhoGDI and restored by the addition of prenylated RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Western blot analysis indicates that this RhoGDI treatment results in the extraction of RhoA from the nuclear envelope. These data support a role for a RhoA-mediated activation of PLD in our recently described hypothesis, which proposes that a signal transduction cascade exists in the nuclear envelope and represents a novel signal transduction cascade that we have termed NEST (nuclear envelope signal transduction).