BACKGROUND: In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays. RESULTS: We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of alpha-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion. CONCLUSION: This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.