Rhotekin, an effector of Rho, is highly expressed in the brain but its function(s) in neurons is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin by yeast two-hybrid screening of human brain cDNA library. We then found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in COS7 cells and the rat brain. The C-terminal three amino acids of Rhotekin were essential for the interaction with Lin-7B. Their binding affinity became increased in the presence of active RhoA in the COS7 cell expression system. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.