Since the identification of pyridine nucleotide Ca2+ mobilising messengers(1), the mechanism by which cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilise intracellular Ca2+ stores has remained controversial. A wealth of evidence across a variety of cell types has suggested that cADPR activates ryanodine receptors (RyRs). However, studies on reconstituted RyRs in lipid bilayers have failed to demonstrate direct regulation of these channels by cADPR(2) and it has even been suggested that cADPR may initiate intracellular Ca2+ release by promoting Ca2+ uptake via the sarco / endoplasmic reticulum Ca2+ ATPase(3). Most recently, we have proposed that NAADP mobilises endolysosomal Ca2+ stores by activating two pore channels (TPC, TPCN for gene name)(4) while others have suggested that NAADP may activate RyRs(5). We therefore assessed (by Fura-2 fluorescence ratio, F340 / F380) Ca2+ signals evoked by intracellular dialysis from a patch-pipette (voltage-clamp mode, -40 mV holding potential) of cADPR (100 μM) and NAADP (10 nM) into HEK293 cells that stably over-express either human TPC2, rabbit RyR1, or mink RyR3. No change in Fura-2 fluorescence ratio was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in HEK 293 cells that stably over-express TPC2 (n = 4). By contrast, a marked and transient increase in the Fura-2 fluorescence ratio was triggered by cADPR in RyR1 expressing cells (from 0.31 ± 0.02 to 0.86 ± 0.13; n = 9) and RyR3 expressing cells (from 0.41 ± 0.04 to 0.76 ± 0.04; n = 5). Our findings with respect to NAADP were almost the exact opposite. Thus, NAADP evoked, as before(5), a Ca2+ transient in TPC2 expressing cells (F340/ F380 increased from 0.36 ± 0.01 to 0.99 ± 0.05; n = 8) but not in cells that expressed RyR3 (F340/ F380 increased from 0.36 ± 0.04 to 0.41 ± 0.07; n = 3), although a relatively small but discernable Ca2+ transient was triggered by NAADP in RyR1 expressing cells (from 0.43 ± 0.07 to 0.67 ± 0.11; n = 3). We conclude, therefore, that cADPR triggers Ca2+ release via RyRs but not via TPC2, while NAADP preferentially activates TPC2.