Expression of heart-type fatty acid binding protein is restricted mainly to the skeletal and cardiac muscles and further regulated by peroxisome proliferator-activated receptor alpha. The molecular basis for the muscle-restricted peroxisome proliferator-activated receptor alpha action on the fatty acid binding gene was analyzed using normal and the receptor-null mice and the cultured cells. Two possible peroxisome proliferator-response elements were found in the promoter region of the mouse gene. A gel shift assay showed that both elements were functional. However, neither the tandem repeats of the elements nor the cloned promoter sequence could be activated by peroxisome proliferator-activated receptor alpha and its ligand in the reporter gene assay using cultured cells. The cloned promoter responded to the ligand only in the muscle when the reporter gene was introduced into the mouse muscle. Using a chimeric receptor with the activation domain of herpes virus VP16 protein and the tandem repeats of the elements with or without mutation, the upstream element was finally demonstrated to be potentially involved in the receptor-dependent transcriptional activation. These results suggest that the peroxisome proliferator-response element of the mouse gene is atypical and there is a muscle-specific mechanism to enhance the weak binding of the receptor to the response element to ensure the muscle-specific action of peroxisome proliferator-activated receptor alpha on the heart-type fatty acid binding protein gene promoter.