Overexpression of COX-2 and enhanced synthesis of PGE₂ have been implicated in human endometrial pathologies. To investigate the molecular role of COX-2, the Ishikawa human endometrial epithelial cell line was stably transfected with the pIRES2 vector containing COX-2 cDNA in either the sense or antisense directions. PGE₂ concentrations were significantly elevated in the cells transfected with the COX-2 sense compared with wild-type cells or cells transfected with the antisense cDNA (P < 0.01). Elevated PGE₂ synthesis was associated with enhanced expression and signaling of EP receptors. cDNA array analysis revealed differential expression of cathepsin D between the COX-2 sense and antisense cells. Cathepsin D RNA and protein expression was 6.7 and 2.1 fold lower in the COX-2 sense compared with COX-2 antisense cells respectively. Cathepsin D is known to cleave plasminogen to the potent anti-angiogenic factor angiostatin. To investigate differential angiostatin generation, conditioned media from COX-2 sense, COX-2 antisense and wild-type cells were incubated with plasminogen and subsequently subjected to Western blot analysis. In comparison to wild-type cells, the cleavage of plasminogen to angiostatin was abolished when incubated in COX-2 sense cells conditioned media and elevated when incubated in COX-2 antisense cells conditioned media. Co-incubation of plasminogen with the cathepsin D inhibitor Pepstatin A inhibited the cleavage of plasminogen to angiostatin in the COX-2 antisense conditioned media. These data demonstrate that COX-2 exerts a negative feedback on the expression of cathepsin D. This in turn reduces the generation of the anti-angiogenic factor angiostatin, hence promoting a pro-angiogenic environment.