The seven-transmembrane receptor Smoothened (Smo) transduces the signal initiated by Hedgehog (Hh) morphogen binding to the receptor Patched (Ptc). We have reinvestigated the pharmacological properties of reference molecules acting on the Hh pathway using various Hh responses and a novel functional assay based on the coexpression of Smo with the alpha subunit of the G15 protein in HEK293 cells. The measurement of inositol phosphate (IP) accumulation shows that Smo has constitutive activity, a response blocked by Ptc which indicates a functional Hh receptor complex. Interestingly, the antagonists cyclopamine, Cur61414, and SANT-1 display inverse agonist properties and the agonist SAG has no effect at the Smo-induced IP response, but converts Ptc-mediated inactive forms of Smo into active ones. An oncogenic Smo mutant does not mediate an increase in IP response, presumably reflecting its inability to reach the cell membrane. These studies identify novel properties of molecules displaying potential interest in the treatment of various cancers and brain diseases, and demonstrate that Smo is capable of signaling through G15.