The two gluconeogenic substrains of renal epithelial cells, LLC-PK1-FBPase+ and OKGNG+, have been shown to differ markedly in their metabolism of lactate and pyruvate. OKGNG+ cells consumed lactate as well as pyruvate at high rates in contrast to LLC-PK1-FBPase+ cells, which failed to take up or utilize lactate. (Aminooxy)acetate (AOA), an inhibitor of transamination reactions, was used to further delineate these differences. Lactate consumption of OKGNG+ cells was significantly inhibited by AOA, whereas pyruvate consumption by LLC-PK1-FBPase+ cells was slightly stimulated. Growth of OKGNG+ cultures, however, could be achieved on lactate in the presence of AOA. From these results it was concluded that the cell strains might differ in the subcellular distribution of phosphoenolpyruvate carboxykinase (PEPCK). LLC-PK1-FBPase+ cells may express both mitochondrial and cytosolic PEPCK isoenzymes, whereas OKGNG+ cells express only the mitochondrial isoenzyme. This was tested by directly assaying PEPCK activity in subcellular fractions of the cells. In OKGNG+ cells PEPCK activity fractionated with the mitochondrial marker glutamate dehydrogenase; however, in LLC-PK1-FBPase+ cells two-thirds of PEPCK activity was found in the cytosol. In LLC-PK1-FBPase+ cells, PEPCK activity increased twofold on incubation in acidic culture medium (pH 6.9) for 18 h, in contrast to the PEPCK activity in OKGNG+ cells. Northern blot analysis using cDNA probes specific for the mitochondrial and cytosolic PEPCK mRNAs confirmed the enzyme activity data. In LLC-PK1-FBPase+ cells strong expression of cytosolic PEPCK mRNA was observed, whereas in OKGNG+ cells only very low levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)