Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent β-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat λDASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBPα (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBPα confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBPα, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.