The transcription of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is differentially regulated in each of the several PEPCK-C-expressing tissues. In the kidney, it is regulated by glucocorticoids and acidosis. Previously, we reported that in LLC-PK1 and derived kidney cell lines, mutation of the hepatic nuclear factor 1 (HNF-1) binding site in PEPCK-C gene promoter markedly reduced both the basal activity of the gene promoter and its response to acidic pH. Using the same kidney cell line, we now report that nuclear receptors robustly stimulate transcription from the PEPCK-C gene promoter. This stimulation is markedly reduced by mutation of the accessory factor 1 (AF1) site in the glucocorticoid responsive unit (GRU) residing within the glucocorticoid-responsive domain. The stimulation is likewise reduced by mutation of the HNF-1 site, residing outside the nuclear receptor-responsive domain of the PEPCK-C gene promoter. There is no binding similarity between HNF-1 and AF1 binding sites, as is evident from gel shift assays showing a lack of competition of either site for the binding of renal nuclear proteins to the other. We further assessed that the regulation of PEPCK-C gene transcription by acidosis is not mediated by nuclear receptors. This became evident from studies of transgenic mice harboring a rat PEPCK-C transgene driven by truncated 5' flanking region of the gene, which contains the HNF-1 site but lacks the glucocorticoid responsive domain. The full transcriptional response of this transgene to acidosis establishes that the truncated 5' flanking region (362 bp) of the PEPCK-C gene contains the information required for the acidosis-mediated regulation independent of the glucocorticoid domain. Taking together the previous and present results, it appears that acidosis and nuclear receptors regulate the renal transcription of the PEPCK-C gene via two independent domains in the 5' flanking region of the gene. These two modulations, as well as the basal activity of the gene, require intact HNF-1 binding site in the gene promoter.