Fibroblast cell lines are frequently used to diagnose genetic mitochondrial defects in children. The effect of enzyme deficiency on overall flux rate through metabolic pathways is, however, not generally considered. We have transposed an experimental paradigm that was developed for isolated perfused organs using (13)C-labeled substrates and (13)C-isotopomer analysis to probe pyruvate mitochondrial metabolism in cultured human fibroblast cell lines with normal or genetically mutant pyruvate decarboxylation (PDC) or carboxylation (PC) activity. Cells were incubated with 1mM [U-(13)C]pyruvate, and the (13)C-molar percent enrichment (MPE) of intracellular pyruvate, citrate, malate (as a surrogate of oxaloacetate) and aspartate was assessed by mass spectrometry. We estimated various flux ratios relevant to metabolic pathways involved in energy production, namely pyruvate formation, PDC, PC, and citrate recycling in the citric acid cycle (CAC). In all cell lines, exogenous pyruvate was predominately decarboxylated (PC/PDC ratios 0.01-0.3). PC-deficient cell lines displayed an expected negligible contribution of PC flux to oxaloacetate formation for citrate synthesis (PC/CS), which was associated with a greater contribution of PDC to acetyl-CoA formation (PDC/CS), and greater recycling of (13)C-labeled citrate into the CAC. In PDH-deficient cell lines, metabolic flux alterations were most apparent in cells with more than 50% reduction in enzyme activity. This led to an unexpected lower PC/CS flux ratio, while the PDC/CS flux ratio was unchanged. These data illustrate the usefulness of this approach in identifying unexpected metabolic consequences of genetic defects related to pyruvate metabolism.