Abstract Cyclooxygenase 2 (COX-2) enzyme, which catalyzes the synthesis of several pro- and anti-tumorigenic prostanoids, is associated with more than 40% of breast cancers. Selective and non-selective COX-2 inhibitors were shown to decrease the risk of tumorigenesis and breast cancer recurrence. However, the precise role of COX-2 in the mammary epithelium, and the potential role of COX-1, remains ill-defined. This is in part because of infertility, renal pathology and short life-span that accompanies global deletion of the COX-2 gene in mice. To study the role and mechanisms of COX-2 actions in breast cancer, we engineered mice on a pure FVB/N background that lack COX-2 only in mammary epithelial cells (MECs). Mice transgenic for COX-2 exons 5 and 6, flanked by loxP sites (COX-2flox/flox), were crossed with mice expressing cre recombinase under control of the mouse mammary tumor virus (mmtv) promoter (Cremmtv) to generated the MEC-targeted deletion of COX-2. MECs harvested from wild type (WT; COX-2flox/flox) and knock-out (KO; Cremmtv COX-2flox/flox) mice were treated, in vitro, with or without a single dose of 5µg/ml lipopolysaccaride (LPS). KO MECs showed about 80% less COX-2 mRNA and protein expression. Prostaglandin (PG) E2 production was similarly reduced in KO MECs, despite unchanged levels of COX-1 mRNA and protein (n=5-7). We confirmed COX-2 as the principle source of PGE2 in MECs using inhibitors specific for COX-2 (rofecoxib) or COX-1 (FR122047) (n=6). Expression of COX-2 and COX-1 mRNA and protein, and PGE2 generation, were not significantly different in untreated or LPS-treated peritoneal macrophages obtained from WT and KO mice (n=5-6) confirming the specificity of the targeted COX-2 deletion to MECs. Unlike global COX-2 deficiency, mice lacking COX-2 in MECs were viable, fertile and disease-free making them highly suitable for cancer studies. To induce breast tumorigenesis, 6 week old virgin female WT and KO mice received medroxyprogesterone acetate implants (75 mg/pellet, 21 day release) subcutaneously followed, at 9 weeks of age, by 1mg/week of 7,12-dimethylbenz[a]anthracene (DMBA) administration for 4 weeks. This regime causes predominantly mammary tumor development in mice. Starting from week 13, mice were checked weekly for mammary tumors. The onset of breast cancer, denoted as the week of age during which a mammary tumor was palpated in the glands of a live mouse, was significantly delayed in KO mice compared to their WT counterparts (p=0.03); the median duration of tumor free period was 28 weeks for KO animals (n=36) versus 22 weeks for WT mice (n=20). These data indicate that targeted deletion of COX-2 in mammary epithelium affords significant protection against initiation and progression of breast tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1602.