Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent intracellular Ca2+-mobilising messenger so far discovered. It is highly unusual in that it appears to release calcium, from acidic calcium stores such as lysosomes. Recently the two-pore channel (TPC) family of ion channels have been identified as the long sought after molecular targets for NAADP [1,2,3]. Little however is known about the molecular nature of these proteins in animal cells. The TPC genes encode a family of proteins with two predicted repeats of six transmembrane domains with a putative pore loop between transmembrane domains five and six in each repeat and several consensus sites for N-glycosylation. This structure is analogous to one half of a voltage-gated calcium channel. It has therefore been proposed that TPCs exist as dimers such that each subunit contributes 2 of the 4 domains required to generate a functional channel. Humans possess two TPC isoforms (HsTPC1-2) whereas sea urchins a model organism for study of NAADP mediated signalling, possess three TPC isoforms (SpTPC1-3). Here, we analyzed HsTPC1 using fluorescence protease protection assays [4]. Our results are consistent with the current topology model. We further show that both human and sea urchin TPC1 and TPC2 (but not TPC3) are N-linked glycosylated, as evidenced by a reduction in size on SDS-PAGE after treatment by PNGase F from a glycosylated size of ~140kDa to the predicted core size of ~95kDa in the case of HsTPC1 (n = 7). Mutation of N599, N611 and N616 abolished glycosylation placing these residues within a luminal loop, again consistent with the predicted topology. Glycosylation did not appear to regulate trafficking however as confocal microscopy shows the localisation of the HsTPC1 glycosylation-defective mutant remains unchanged. Finally, we determined the native molecular weight of TPCs. Whereas sucrose density gradient fractionation indicated that TPCs migrated as dimers (mw ~ 200 kDa), results from gel filtration analysis indicated a possible tetrameric structure (mw ~ 400 kDa). This aberrant mobility is consistent with previous raioligand binding studies of NAADP receptors from sea urchin egg homogenates [5]. Our data provide new molecular insight into this newly discovered family of channels in NAADP mediated signalling.