The present study was performed to assess the influence of IL-1β and/or camptothecin (CPT-11) on nitric oxide (NO) secretion by co-cultures of 3 human colon adenocarcinoma cell lines: HT29, LS180 and SW948, derived from different grades of tumours (Duke’s grade) cultivated as spheroids with normal human colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Tumour cell spheroids in monoculture produced higher amounts of NO than normal cells. In co-cultures, the level of the radical decreased compared to the sum of NO produced by tumour and normal cell monocultures. IL-1β non-significantly induced NO production in colon tumour cell spheroids and normal cell monolayers, but significantly induced the radical production in co-culture of low grade HT29 tumour cell spheroids with normal cells. CPT-11 used alone limited NO production, while in combination with IL-1β it increased the level of the radical. IL-1β and CPT-11, dependent on whether they are added separately or jointly, differentially modulate NO in monocultures of colon tumour spheroids or normal cells and their co-cultures.