Pepsinogen C is an aspartic proteinase mainly involved in the digestion of proteins in the stomach, which is also synthesized by certain human breast tumors. To examine the possibility that extragastric production of this proteolytic enzyme could be mediated by hormonal factors, we have analyzed pepsinogen C gene expression in human breast cancer cells subjected to different hormonal treatments. Northern blot analyses revealed the expression of pepsinogen C gene by T-47D breast cancer cells after induction with dihydrotestosterone, dexamethasone, and progesterone but not with estradiol, retinoic acid, or ethanol. Reverse transcription-polymerase chain reaction analysis in a series of breast cancer cell lines confirmed the amplification of pepsinogen C mRNA after induction with dihydrotestosterone, in those cells expressing the androgen receptor mRNA. The promoter region of the pepsinogen C gene was functionally characterized by transient expression of a vector containing the promoter region cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene. CAT activity in T-47D cells was stimulated in the presence of dihydrotestosterone, dexamethasone, and progesterone but not by estradiol. By further deletion mapping of the pepsinogen C promoter, a minimal region (AGAACTattTGTTCC) was identified as being responsible for glucocorticoid-, androgen-, and progesterone-regulated gene expression.