The characteristics of a cis-acting regulatory region involved in the human-aromatase P-450 gene have been examined by transient expression analysis. The region spans from -242 - -166 relative to the cap site of the gene. A fragment containing the region excised from the gene enhances heterologous promoter activity as well as its own promoter activity in a position-independent and orientation-independent manner. The fragment exerts its enhancer activity in human BeWo choriocarcinoma cells in which the aromatase P-450 gene is expressed, but not in other cell lines tested. Deletion of 38 bp from the 3' end of the fragment results in a complete loss of enhancer activity. A gel-retardation assay with nuclear extracts from BeWo cells suggests the existence of a nuclear factor(s) which interacts with the fragment. These results suggest that the regulatory element in the fragment is involved in efficient transcription of the human-aromatase P-450 gene.