To determine the effect of nitric oxide (NO) on the cyclooxygenase-2 (COX-2) regulation in colon cancer cell lines we used the physiological NO donor GSNO. The proximal 6.6 kb of the COX-2 promoter was cloned into the pGL3 basic vector and the sequential deletion of the 6.6 kb COX-2 promoter generated promoter constructs of 4, 2.6, 1.9 and 0.9 kb. These constructs clearly show that the main regulatory region lies within 0.9 kb of the transcription start site. Therefore, constructs of the main transcription binding sites within this region namely CRE, NF-IL6 and NF-kappaB and mutations of these sites were used to monitor the transcriptional activation of COX-2. This study was performed on the colon cancer cell lines HCA7 and HCT116 which have a differential expression of COX-2. There was no evidence that the luciferase activity is negatively affected by NO as was previously reported. The CRE and NF-IL6 binding sites within this region were responsible for the constitutive and physiological NO-induced COX-2 transcriptional activity in the HCA7 and HCT116 cells. While NF-kappaB involvement was only observed in the HCT116 cells, the cell lines displayed increased NF-kappaB transcriptional activity after exposure to NO.