Transcription of mitochondrial serine:pyruvate aminotransferase (SPT) mRNA (SPTm-mRNA) in rat liver is unique in that it occurs from the upstream site of the two transcription start sites within the first exon of the SPT gene and is selectively enhanced by cAMP via the protein kinase A (PKA) signaling pathway. In this study, we identified the DNA elements and nuclear factors responsible for the basal and PKA-induced activities of the upstream promoter. By using a luciferase reporter assay with HepG2 cells, DNase I footprinting analysis, and gel shift experiments, we identified the binding sites for Sp1 and AP-2 within the regions -125 to -89 and -14 to +10, respectively. Mutational analyses indicated that these regions are essential for the transcription factor binding and the SPT promoter activity. Expression of AP-2 caused a marked increase in the basal promoter activity to about the same level as that achieved by PKA. On the other hand, both the basal and PKA-induced activities were elevated by overexpression of Sp1, its effect on PKA-induced activity being more pronounced with coexpression of CBP and repressed by E1A oncoprotein. These results suggest that AP-2 and Sp1 regulate basal promoter activity, and Sp1 is also involved in PKA-mediated expression of the rat SPT gene in concert with the transcriptional coactivator CBP.