In order to compare the trafficking of proteins with different membrane anchors, we have constructed and expressed three different recombinant forms of neutral endopeptidase (NEP) in MDCK cells. The wild type form of NEP (WT-NEP) is attached to the plasma membrane by a single N-terminal membrane spanning domain, whereas the glycosylphosphatidylinositol-anchored form of the protein (GPI-NEP) contains a C-terminal GPI anchor. A double anchored form of NEP (DA-NEP) was also constructed, that contains both the original N-terminal membrane spanning domain and a C-terminal GPI anchor. We show here that WT-NEP, GPI-NEP and DA-NEP, which are all apically targeted in MDCK cells, behave differently when subjected to Triton X-100 solubilisation: despite the presence of the transmembrane anchor DA-NEP behaves as a GPI-anchored protein. This suggests that the GPI anchor of DA-NEP is dominant over the transmembrane anchor of the native protein to determine its pattern of solubility in Triton X-100.