We investigated the consequences of depolarizing the mitochondrial membrane potential (∆ψ_mit) on Ca²⁺ signals arising via inositol 1,4,5-trisphosphate receptors (InsP₃R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize ∆ψ_mit. Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca²⁺-mobilizing hormone and prolonged the recovery of cytosolic Ca²⁺ after washout of the hormone; effects consistent with the removal of a passive Ca²⁺ buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca²⁺ changes were observed to progressively decline. The inhibition of Ca²⁺ signalling was observed using different Ca²⁺-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P₃ ester. Upon washout of FCCP, the Ca²⁺ signals recovered with a time course similar to the re-establishment of ∆ψ_mit. Global measurements indicated that none of the obvious factors such as changes in pH, ATP concentration, cellular redox state, permeability transition pore activation or reduction in Ca²⁺-store loading appeared to underlie the inhibition of Ca²⁺ signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing ∆ψ_mit, prevents InsP₃R activation.