CYP3A16 is expressed in the mouse liver specifically during a fetal and puberty life. Functional regions responsible for the transcriptional regulation of the Cyp3a16 gene were identified by an in vivo direct DNA injection into mouse livers followed by a luciferase assay. The results of the deletion analysis of a 5'-flanking sequence suggested the existence of an adult-specific repressor(s) interacting with the Cyp3a16 gene. In addition, a positive regulatory element was assumed to be present in a region from -146 to -56 of the Cyp3a16 gene. A hepatocyte nuclear factor 4 (HNF-4)-binding consensus sequence was found in this region. In fact, HNF-4 alpha bound to this sequence as evidenced by a gel mobility shift assay. The role of the HNF-4-binding sequence was further examined by an introduction of mutations in this sequence. The introduction of the mutations resulted in a reduced activity of a luciferase in the assay. These results obtained by an application of the direct DNA injection method suggest that the HNF-4 alpha activates the transcription of the Cyp3a16 gene in vivo.