The present study investigated the activity and cellular localization of individual isoenzymes of nitric oxide synthase (NOS) using immunohistochemistry and the [3H]citrulline assay in normal colorectal epithelia and neoplastic tissue. Intracellular localization of isoenzymes of NOS was detectable by immunohistochemistry in normal epithelial cells. Colorectal adenocarcinomas had a marked reduction of both Inducible NOS (iNOS) and constitutive NOS (cNOS) expression. Expression of iNOS was completely absent in tumour cells (P < 0.0001), while cNOS was reduced in 66% and absent in 34% of tumours studied when compared with controls (P < 0.0001). NOS activity using the [3H]citrulline assay was detectable in normal epithelium and was found to be reduced in tumours (P < 0.001). In addition, colonic adenomas had reduced INOS but not cNOS expression when compared with controls (P < 0.003 and P = 0.39 respectively). We conclude that NOS is present and active within the epithelium of the normal colon, with localization of the individual isoenzymes. Furthermore, there was loss of activity and expression of individual isoenzymes in colonic neoplasms.