GS32/SNAP-29 is a SNAP-25-like SNARE and has been shown to interact with syntaxin 6. Using immobilized recombinant GS32, we have recovered EHD1 as a major GS32-interacting protein from total HeLa cell extracts. This interaction is mediated by the EH domain of EHD1 and the N-terminal NPF-containing 17-residue region of GS32. Co-immunoprecipitation suggests that GS32 could also interact with EHD1 in intact cells. When immobilized GST-EHD1 was used to fish out interacting proteins from total brain extracts, syndapin II was identified as a major interacting partner. Similar to the GS32-EHD1 interaction, syndapin II also interacts with the EH domain of EHD1 via its NPF repeat region. Interaction of endogenous EHD1 and syndapin II was also established by co-immunoprecipitation. Furthermore, interaction of GS32 and syndapin II with EHD1 was shown to be mutually exclusive, suggesting that EHD1 may regulate/participate in the functional pathways of both GS32 and syndapin II in a mutual exclusive manner. Opposing roles of GS32 and syndapin II in regulating the surface level of transferrin receptor (TfR) were observed.