Recent studies suggest that adhesion-related kinase (Ark) plays a role in gonadotropin-releasing hormone (GnRH) neuronal physiology. Ark promotes migration of GnRH neurons via Rac GTPase and concomitantly suppresses GnRH gene expression via homeodomain and myocyte enhancer factor-2 (MEF2) transcription factors. Here, we investigated the signaling cascade required for Ark inhibition of the GnRH promoter in GT1-7 GnRH neuronal cells. Ark repression was blocked by the MEK/ERK pathway inhibitor, PD98059, and dominant negative MEK1 but was unaffected by dominant negative Ras. Inhibitors of the Rho family GTPases, Clostridium difficile toxin B (Rho/Rac/Cdc42 inhibitor) and Clostridium sordellii lethal toxin (Rac/Cdc42 inhibitor), blocked Ark inhibition of GnRH transcription. Moreover, dominant negative Rac blunted both Ark activation of ERK and repression of the GnRH promoter, demonstrating an essential role for Rac in coupling Ark to ERK activation. Like Ark, a constitutively active mutant of Rac suppressed GnRH transcription in an ERK-dependent manner. Finally, Ark-mediated repression was significantly attenuated by a dominant negative MEF2C, whereas repression induced by constitutively active Rac was unaffected, indicating that MEF2 proteins are not targets of the Ark --> Rac --> MEK --> ERK cascade. The data suggest that Ark suppresses GnRH gene expression via the coordinated activation of a Rac --> ERK signaling pathway and a distinct MEF2- dependent mechanism.