Calcium is released from intracellular stores by calcium-mobilising messengers inositol trisphosphate (IP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (Patel 2004). Unlike IP3 and cADPR, which release calcium from the endoplasmic reticulum, NAADP is highly unusual releasing calcium from acidic organelles (Churchill et al. 2002). Recently, the two-pore channels (TPCs) have been localized to the endo-lysosomal system and shown to release calcium in response to NAADP (Brailoiu et al. 2009; Calcraft et al. 2009). Little however is known concerning how they are trafficked. Here, we identify conserved di-leucine based targeting motifs in vertebrate TPCs. Confocal microscopy analysis of heterologously expressed GFP constructs revealed that human TPC2 lacking a di-leucine motif in its N-terminus resulted in its relocation to the plasma membrane (n=6). In contrast, removal of the N-terminus from TPC1 (n=4), mutation of second di-leucine motif near the C-terminus (n=4) or combining both modifications (n=5), resulted in the expressed proteins retaining an intracellular distribution similar to that of wild-type TPC1 (n=5). These results suggest that the di-leucine motif present in TPC2 is critical for its trafficking to the endo-lysosomal system but that other signals are required for TPC1. We thus provide the first molecular insight into targeting of this novel ion channel family.