LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.