Netrin-1 is a pleiotropic signaling molecule first discovered for its role in neuronal development, where it is largely responsible for axonal guidance. When signaling through the DCC receptor, netrin-1 serves as a chemoattractant; however, signaling through the UNC5 receptor results in chemorepulsive activity (Ko et al., 2012). In the free-living ciliate, Tetrahymena thermophila, netrin-1 and netrin-1 peptide act as chemorepellents at micromolar to nanomolar concentrations, causing cells to exhibit avoidance behavior. While many pharmacological inhibitors that we tested had no effect on avoidance behavior, the tyrosine kinase inhibitor, genistein (IC50 ~ 50 μg/ml), inhibited avoidance behavior in this organism. However, when using the monoclonal anti-phosphotyrosine antibody PT-66 to probe for phosphotyrosine in control and netrin-1 exposed cells, both control and netrin-1 exposed cells showed a low level of immunostaining, indicating that tyrosine phosphorylation was not required for netrin signaling. The localization of the staining was also similar in both groups. Genomic studies of Tetrahymena thermophila (Eisen et al., 2006) indicate that no tyrosine kinases are found in this organism. Previous biochemical studies (Christensen et al., 2003; Bartholomew et al., 2008) have suggested that tyrosine kinases are required for signaling in this organism. Our current data imply that the tyrosine kinase inhibitors we used may be binding to an alternative site in Tetrahymena. Additional studies will be necessary in order to determine the mechanism by which tyrosine kinase inhibitors are blocking netrin-1 avoidance in this organism.