The gene encoding the mammalian two-pore channel 1 (tpc1) is homologous to the Arabidopsis thaliana, tpc gene, which encodes a class of Ca²⁺-dependent Ca²⁺-release channels present in the vacuolar membrane (Maathuis et al. 2005). tpc1 is expressed in epithelial tissues including the kidney where it is expressed in the collecting duct (Ishibashi et al. 2000). In order to investigate the possible physiological functions of tpc1 in renal collecting duct cells we have determined the expression and sub-cellular location of tpc1 in mouse renal collecting duct cell-lines (M1, mIMCD-K2 and mIMCD-3). Using gene-specific primers for murine tpc1 we have detected expression of tpc1 in all 3 cell-lines by RT-PCR of a 450bp PCR product confirmed by direct sequencing. Using IMAGE clone 6821376 as template, tpc1 was cloned into expression vector pcDNA3.1/NT-GFP producing a tpc1/N-terminal GFP construct (tpc1-NT-GFP). Sub-cellular localisation of tpc1-NT-GFP was determined after Lipofectamine-mediated transfection of sub-confluent mIMCD-3 cells grown on coverslips. Cells were imaged 24-48 h post-transfection, using confocal microscopy as previously described (Sayer et al. 2001). Alternatively a TPC1 antibody raised against a C-terminal peptide AAQQTPGSRQRSQTVT was used to localise mtpc1 by immunohistochemistry on filter grown collecting duct cell-lines. In mIMCD-3 cells tpc1-NT-GFP localised to the plasma membrane and intracellular vesicles. Plasma membrane expression was confirmed by brief (2 min) incubation with TRITC conjugated wheat germ lectin prior to methanol fixation and imaging. The nature of the vesicular compartment was further investigated in mIMCD-3 cells by counter staining with Lysotracker Red (marker of acidic endosomes) and by cotransfection with an mCLC-5-CT-RFP fusion (marker of recycling early endosomes) or pDsRED-ER (endoplasmic reticulum marker). Whereas there was minimal overlap with Lysotracker Red or mCLC-5-CT-RFP, there was substantial co-localisation of tpc1-NT-GFP with the endoplasmic reticulum marker. In all 3 cell-lines immunohistochemistry revealed prominent sub-apical vesicular staining. We conclude that the location of tpc1 is consistent with its possible role as a Ca²⁺-influx pathway and as a Ca²⁺-release channel for Ca²⁺ stores in epithelial tissue including the mammalian collecting duct.