The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the critical role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous types of cancer. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the seven-transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a large microtubule (MT) associated multi-protein signaling complex, termed the Hedgehog Signaling Complex (HSC). At a minimum, the HSC consists of the transcription factor Cubitus interruptus (Ci), the protein kinase Fused (Fu) and the kinesin related protein (KRP) Costal2 (Cos2). In response to activated Smo, Cos2 is phosphorylated, releasing the HSC from MTs. This MT release is thought to alter the ratio between repressor (Ci75) and activator forms of Ci, ultimately determining the level of expression of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. We have provided evidence that Smo may directly communicate with Cos2 to regulate HSC function. We suggest a model in which there are two distinct HSCs with discrete sub-cellular Iocalizations and activities: one is endosome associated and facilitates Ci75 production (HSC-R), and one is Smo associated and promotes Ci activation (HSC-A). In response to Hh, and through direct association with Cos2, Smo mediates both inhibition of the endosome associated HSC-R, and activation of HSC-A at the plasma membrane. The experiments proposed here will test this model. We will identify three distinct domains on Cos2, which associate with MT, endosomes, or with Smo. We will then demonstrate that these domains are necessary and sufficient for localizing the HSC to various sub-cellular locations. We will then show Fu regulates the ability of Cos2 to redistribute Ci in response to Hh. Finally, we will test this model in vivo, using these various Cos2 mutants to selectively activate HSC-A or inhibit HSC-R.