Arrhythmogenic point mutations in RyR2 result in abnormal Ca2+ release following cardiac stimulation, leading to sudden cardiac death (SCD). Recently, we have demonstrated that significant functional differences exist between SCD-linked RyR2 mutations. Here, we investigated the molecular basis of this heterogeneity and determined the sensitivity of mutant RyR2 channels to cytoplasmic [Ca2+] ([Ca2+](c)) in living cells. Using streptolysin-O permeabilised human embryonic kidney cells, [Ca2+], was clamped in cells expressing GFP-tagged wild-type (WT) or SCD-linked RyR2 mutants ((LP)-P-433, (NI)-I-2386, and R(176)Q/(TM)-M-2504). Although resting [Ca2+](c) was comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca2+-dependent inhibition following caffeine stimulation when compared with WT channels. The ER Ca2+ store was not perturbed in these experiments. Our findings support the hypothesis that SCD-linked mutational loci may be an important mechanistic determinant of RyR2 dysfunction and indicate that there is unlikely to be a unifying mechanism for channel dysfunction in SCD. © 2005 Elsevier Inc. All rights reserved.