The effect of the cytoskeletal protein zyxin on the Hedgehog (Hh) signaling pathway was demonstrated in our earlier study on the role of zyxin in early patterning of the central nervous system (CNS) anlage of Xenopus laevis. The present study demonstrates that zyxin can physically interact with Patched2 (Ptc2), the transmembrane Hh receptor. This ability has been confirmed by a series of experiments on the immunoprecipitation of the zyxin-Ptc2 complex and the complexes formed by the deletion mutants of these proteins. It has been shown that the interaction between zyxin and Ptc2 is determined by the ability of the second LIM domain of zyxin (amino acid residues 530-590) to interact with the fragment of the C-terminal cytoplasmic juxtamembrane region of the X. laevis Ptc2 (residues 1220-1312). We have also demonstrated the interaction between the homologous domains of human zyxin and Ptc1, the human Hh receptor. The obtained data suggest the existence of an evolutionary conserved mechanism for the regulation of the Hh signaling pathway via the interaction between zyxin and the Hh receptor, Ptc.