Aims: Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by β-glucuronidase may represent a significant pathway of SN-38 formation. Methods: The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 μ m) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). Results: The rate of conversion of irinotecan (9.6 μ m) was lower in tumour tissue than matched normal colon mucosa samples (0.30 ± 0.14 pmol min-1 mg-1 protein and 0.77 ± 0.59 pmol min -1 mg-1 protein, respectively; P < 0.005). In contrast, no significant difference was observed in β-glucuronidase activity between tumour and matched normal colon samples (4.56 ± 6.9 pmol min -1 mg-1 protein and 3.62 ± 2.95 pmol min -1 mg-1 protein, respectively, using 9.6 μ m SN-38 glucuronide; P > 0.05). β-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2 > 0.23, P < 0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of β-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P < 0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. Conclusions: Tumour β-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo.