In human, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible of numerous pathologies. However, signalling mechanisms involving these receptors are less well characterised in mammals than in drosophila. To obtain structure-function relationships information on human Patched and Smoothened, we expressed these human receptors in drosophila Schneider 2 cells. We show here that, as its drosophila counterpart, human Patched is able to repress the signalling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in drosophila cells. We also provide experiments showing that human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine suggesting that it is expressed in a native conformational state. Furthermore, contrary to drosophila Smoothened , human Smoothened does not interact with the kinesin Costal 2, and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labelling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in drosophila cells where it undergoes a regulation of its localisation comparable to its drosophila homologue. Thus, we propose that the upstream part of Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched and Smoothened enrichment at the plasma membrane is highly conserved between drosophila and human ; in contrast, signalling downstream of Smoothened is different.