Sequence analysis using the Promoser program predicted two promoter-like regions for rat mtGPAT: a distal promoter approximately 30kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8- and 4.8-fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5' RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7-fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, the results suggest that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.