Although the cGMP/cGMP-dependent protein kinase (cGK) signaling is involved in the regulation of neurite outgrowth, its mechanism remains to be clarified. In this study, we identified a Rho effector, rhotekin, as a cGK-I-interacting protein. Rhotekin was also a substrate for cGK-I alpha. In neurite-extended Neuro2A neuroblastoma cells, cGK-1 alpha and rhotekin were colocalized in the plasma membrane and extended neurites, while treatment with cGMP resulted in translocation of rhotekin to the cytoplasm. In addition, we found that cGK-1 alpha and rhotekin synergistically suppressed Rho-induced neurite retraction. Our findings suggest that cGK-I alpha interacts with and phosphorylates rhotekin, thereby contributing to neurite outgrowth regulation. (C) 2012 Elsevier Inc. All rights reserved.