The activity of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (PEPCK), a rate-limiting gluconeogenic enzyme, was found decreased by others in genetically determined disorders and in Sudden Infant Death Syndrome (SIDS). To understand these findings, we made a systematic study of normal human hepatic PEPCK activities in specimens obtained under various conditions from patients not suspected of having SIDS. PEPCK was assayed by the method of Ballard and Hanson [J. Biol. Chem., 244 (1969) 5625] and activity reported as units (1 mumol/min) per gram protein. Intra-assay precision was 4.1% (n = 1094); inter-assay precision using the same homogenate was 10.4% (n = 51); and inter-assay precision using different homogenates of the same tissue specimen was 16.3% (n = 17). The assay was linear with time and enzyme concentration for at least 60 min up to 1.3 mU/assay and for at least 5 min up to 20 mU/assay. Biopsy specimens had significantly (P = 0.015) higher PEPCK activity, 12.60 +/- 3.01 U/g (range 3.5-10.4, n = 9) compared to specimens obtained at autopsy, 3.20 +/- 0.45 U/g (range 0-8.6, n = 33). Specific activity was not significantly correlated with the patient's age, fresh vs. frozen tissue, postmortem intervals up to 68 h, or length of storage at -70 degrees C up to 21 years. One patient had activity at autopsy (tissue obtained less than 2 h postmortem) 26% less than was observed in his biopsy specimen. Autopsy samples separated by differential centrifugation into mitochondrial and cytosolic fractions and checked with marker enzymes ornithine transcarbamylase (mitochondrial) and arginase (cytosolic) had considerable cross-contamination between the two fractions in fresh and frozen specimens.