The homeodomain-containing transcription factor NKX3.1 is a putative prostate tumor suppressor that is expressed in a largely prostate-specific and androgen-regulated manner. Loss of NKX3.1 protein expression is common in human prostate carcinomas and prostatic intraepithelial neoplasia (PIN) lesions and correlates with tumor progression. Disruption of the murine Nkx3.1 gene results in defects in prostate branching morphogenesis, secretions, and growth. To more closely mimic the pattern of NKX3.1 loss that occurs in human prostate tumors, we have used Cre-and loxP-mediated recombination to delete the Nkx3.1 gene in the prostates of adult transgenic mice. Conditional deletion of one or both alleles of Nkx3.1 leads to the development of preinvasive lesions that resemble PIN. The pattern of expression of several biomarkers (Ki-67, E-cadherin, and high-molecular-weight cytokeratins) in these PIN lesions resembled that observed in human cases of PIN. Furthermore, PIN foci in mice with conditional deletion of a single Nkx3.1 allele lose expression of the wild-type allele. Our results support the role of NKX3.1 as a prostate tumor suppressor and indicate a role for this gene in tumor initiation.