Loss of Nkx3.1 function in mice results in defects in prostate development and epithelial hyperplasia, indicating that this gene plays important roles in both the initiation and maintenance of prostate differentiation. In humans, decreased NKX3.1 expression is associated with the progression of prostate cancer. Despite these roles in prostate development and disease, the transcriptional regulation of Nkx3.1 has not been systematically addressed. A reporter gene approach in transgenic mice was used to identify regulatory regions that dictate the expression pattern of Nkx3.1. A 32-kb DNA fragment from the Nkx3.1 locus that specifies the expected expression pattern during embryogenesis and postnatal life has been identified. Deletion analyses demonstrated that cis-regulatory elements that mediate expression in distinct sites are separable. A 5-kb fragment downstream of the Nkx3.1 coding region contains elements that support expression in the prostate and bulbourethral glands, whereas an upstream fragment contains elements that direct expression in somites and testes. Reporter gene expression analyses also revealed several previously unknown sites of Nkx3.1 expression in males, including urethral glands, glandular cells in the urethral diverticulum and basal epithelial cells in the prostate. In addition, these analyses revealed Nkx3.1 expression in female urethral glands. The identification of Nkx3.1 cis-regulatory elements provides a unique starting point to dissect signaling pathways involved in prostate organogenesis and pathogenesis and provides a system to perturb gene expression throughout prostate development. Developmental Dynamics 234:961–973, 2005. © 2005 Wiley-Liss, Inc.