Physiological and pathological Ca²⁺ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca²⁺ into mitochondria. Mobilization of Ca²⁺ stores with bradykinin (BK) increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca²⁺-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca²⁺ concentration ([Ca²⁺]_mt) of 2.0 ± 0.2 µM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca²⁺]_mt increase, although it did not affect the [Ca²⁺]_c transient. Basal [Ca²⁺]_c and [Ca²⁺]_mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited [Ca²⁺]_mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca²⁺. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca²⁺.