The store-operated calcium-release-activated calcium current, I (CRAC), is a major mechanism for calcium entry into non-excitable cells. I (CRAC) refills calcium stores and permits sustained calcium signalling. The relationship between inositol 1,4,5-trisphosphate receptor (InsP(3)R)-containing stores and I (CRAC) is not understood. A model of global InsP(3)R store depletion coupling with I (CRAC) activation may be simplistic, since intracellular stores are heterogeneous in their release and refilling activities. Here we use a ligand-gated calcium channel, TRPV1 (transient receptor potential channel, vanilloid subfamily member 1), as a new tool to probe store heterogeneity and define intracellular calcium compartments in a mast cell line. TRPV1 has activity as an intracellular release channel but does not mediate global calcium store depletion and does not invade a store coupled with I (CRAC). Intracellular TRPV1 localizes to a subset of the InsP(3)R-containing stores. TRPV1 sensitivity functionally subdivides the InsP(3)-sensitive store, as does heterogeneity in the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms responsible for store refilling. These results provide unequivocal evidence that a specific 'CRAC store' exists within the InsP(3)-releasable calcium stores and describe a novel methodology for manipulation of intracellular free calcium.