Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides, required for DNA synthesis, and is also a critical target for fluoropyrimidines, which are widely used in the treatment of gastrointestinal tumours. We examined TS expression in tumours from 86 patients with advanced colorectal cancer who received one of two chemotherapy regimes (either irinotecan alone or irinotecan and 5-flurouracil with folinic acid). TS expression was determined immunohistochemically in 86 paraffin-embedded primary tumour sections and assessed using image analysis software. TS was significantly associated with survival, with lower levels of TS expression associated with longer patient survival (p=0.02). Patients with an objective response to treatment had a longer median survival than those who did not show an objective response to treatment (p=0.001). A significant association was found between tumour TS expression and response to treatment with 5-FU plus FA with irinotecan (p=0.05). Sixty-four percent of patients with TS expression levels below the median showed an objective (complete or partial) response to treatment while, 38% with TS expression levels above the median responded. Immunohistochemical TS expression levels might be used both as a prognostic marker and to help identify patients who would benefit from 5-FU based regime.