A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO2-) formation. Reverse transcription-polymerase chain reaction (RT-PCR), revealed that all lines (SW480, SW620, DLD-1 and WiDr) expressed mRNA for the Ca(2+)-dependent endothelial (e)NOS, while SW480 cells also expressed the Ca(2+)-dependent neuronal (n)NOS. The mRNA for the Ca(2+)-independent inducible (i)NOS was expressed both by cytokine-stimulated and by unstimulated SW480, SW620 and DLD-1 cells, but none was seen at any time in the WiDr cells. There was, however, little correlation between mRNA expression and enzymic activity based on citrulline and NO2- formation. Thus none of the cell lines exhibited measurable Ca(2+)-dependent NOS activity, while Ca(2+)-independent NOS activity was seen in all but the WiDr cells. Furthermore, DLD-1 cells generated citrulline with resultant NO2- formation only after stimulation with lipopolysaccharide (LPS) and/or cytokines, while SW480 and SW620 did so constitutively. Thus RT-PCR studies indicate that tumour cells of similar epithelial origin display a diverse pattern of NOS gene family expression, and parallel biochemical studies clearly indicate that such expression does not always result in measurable enzymic activity leading to the generation of NO.