The anticancer drug irinotecan (CPT-11) is activated to the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin), by esterases. SN-38 is in turn conjugated to the inactive SN-38 glucuronide (SN-38G). The reverse reaction is mediated by beta-glucuronidases. Hence, production of SN-38 may occur through either pathway. In this study we conducted in vitro studies to examine these two reactions in neuroblastoma xenograft tumors (NB1691) and compared the rates of SN-38 production with those observed in the liver and plasma of the host SCID (severe-combined immunodeficient) mice. The rate of formation of SN-38 from CPT-11 by esterases slowed considerably during a 60-min incubation, consistent with the known deacylation-limited nature of this reaction. For xenograft tumor tissue, K(m) and V(max) values of 1.6 microM and 4.4 pmol/min/mg of protein, respectively, were observed. By comparison, these parameters were estimated to be 6.9 microM and 9.4 pmol/min/mg for mouse liver and 2.1 microM and 40.0 pmol/min/mg for mouse plasma, respectively. The formation of SN-38 from SN-38G was very pronounced in both liver and xenograft tumor tissue, in which it was nonsaturable (0.125-50 microM) and time-independent (0-60 min). The derived values of V(max)/K(m) were 0.65 microl/min/mg for the tumor and 2.12 microl/min/mg for the liver preparations. Microdialysate experiments revealed the concentrations of SN-38G and CPT-11 in tumor to be comparable. At equal substrate concentrations, production of SN-38 from SN-38G in tumor extracts was comparable with that from CPT-11. Therefore, reactivation of SN-38 in the tumor by beta-glucuronidases may represent an important route of tumor drug activation for CPT-11.