Cyclo-oxygenase-2 (COX-2) has important functions in many diseases. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel repressor element in the mouse COX-2 promoter and characterize some of its binding proteins. In order to identify the repressor element, the activity of the mouse COX-2 promoter was investigated in the pancreatic beta-cell line RINm5F using a series of deletion and mutant constructs. The ability of nuclear proteins to bind to this repressor element was then determined by an electrophoretic mobility shift assay and the proteins binding to this repressor element were purified and identified by mass spectrometry. One of the nuclear proteins identified was overexpressed to examine its inhibitory effect on COX-2 promoter activity. We found a novel repressor element located from nucleotides -655 to -632 of the mouse COX-2 promoter region. Some proteins from RINm5F cell nuclear extracts bound to this element, one of which was identified as non-POU-domain-containing, octamer-binding protein (NonO). Overexpression of NonO significantly inhibited wild-type COX-2 promoter activity, but had no effect when the repressor element was mutated. In conclusion, we have demonstrated that a regulatory 'spot' is present in the COX-2 promoter. This provides additional data on COX-2 gene regulation and may provide an insight into the clinical treatment of diseases where COX-2 is highly expressed.