We investigated the consequences of depolarizing the mitochondrial membrane potential (Deltapsi(mit)) on Ca(2+) signals arising via inositol 1,4,5-trisphosphate receptors (InsP(3)R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize Deltapsi(mit). Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca(2+)-mobilizing hormone and prolonged the recovery of cytosolic Ca(2+) after washout of the hormone; effects consistent with the removal of a passive Ca(2+) buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca(2+) changes were observed to progressively decline. The inhibition of Ca(2+) signalling was observed using different Ca(2+)-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P(3) ester. Upon washout of FCCP, the Ca(2+) signals recovered with a time course similar to the re-establishment of Deltapsi(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 Ca(2+)-store loading appeared to underlie the inhibition of Ca(2+) signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing Deltapsi(mit), prevents InsP(3)R activation.