Hoxb13 is robustly transcribed in derivatives of posterior endoderm including the colon, rectum, and the prostate gland. Transcriptional activity in the prostate persists unabated under conditions of androgen deprivation and throughout the course of disease progression in a mouse prostate cancer model. To elucidate the molecular basis of prostate-restricted transcriptional activation of Hoxb13 , a bacterial artificial chromosome (BAC)-based reporter gene deletion analysis was performed in transgenic mice. Two regions downstream of the Hoxb13 coding region were found to be required to support transcriptional activity in the prostate but were completely dispensable for expression in the colon and rectum. Bioinformatic analyses of one region identified a 37-bp element conserved in mammals. This element, which bears two potential binding sites for Forkhead class transcription factors, is occupied by FOXA1 in a human prostate cancer cell line. Precise replacement of this enhancer with an extended LoxP site in the context of a 218,555-bp BAC reporter nearly extinguished Hoxb13 -mediated transcriptional activity in the mouse prostate. These data demonstrate that FOXA1 directly regulates HOXB13 in human prostate epithelial cells, and show that this prostate-specific regulatory mechanism is conserved in mice.