Serum apolipoprotein A(1) (apoA(1)) concentration is inversely correlated with the risk of premature atherosclerosis. Serum apoA(1) concentrations are regulated, in part, at the transcriptional level. ApoA(1) mRNA is synthesized primarily in the liver and small intestine, under the direction of a number of signaling molecules and tissue-specific regulatory elements. Previously, we demonstrated that extracellular acidosis suppresses apoA(1) mRNA levels at the level of transcription. Here we demonstrate that intracellular acidosis, in the absence of extracellular pH changes, represses apoA(1) promoter activity. Repression occurs through a pH responsive element (pH-RE) located within the apoA(1) gene promoter. Acidosis increases the specific DNA binding activity of a putative repressor protein within the immediate 5'-flanking region of the apoA(1) gene. The cis-element that binds the putative repressor protein contains a negative thyroid hormone response element (nTRE) located 3' and adjacent to the apoA(1) TATA box. Mutation of the nTRE/pH-RE abrogates protein binding and alters the activity of reporter genes controlled by this element. Repression by acidosis did not require de novo mRNA and protein synthesis. Inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C (PKC) signaling pathways with tyrophostin A47 and phorbol myristate acetate, respectively, did not affect the repression of apoA(1) promoter activity with acidosis. These results suggest that transcriptional repression of the apoA(1) gene by alterations in ambient pH is associated with enhanced DNA binding activity of a repressor protein, through a mechanism which appears to be independent of de novo mRNA and protein synthesis, tyrosine kinase activity, or PKC activation.