The anticancer agent irinotecan (CPT-11) is a prodrug converted to its active form, SN-38, by human carboxylesterase (hCE) and the SN-38 is further metabolized to its inactive form, SN-38G. We investigated the expression of hCE in human lung cancer cells as well as the ability of these cells to convert CPT-11 to SN-38 using surgically resected tumor samples and cultured cell lines. SN-38 was 40- to 3,000-fold more toxic to lung cancer cell lines than CPT-11, which acted more time-dependently than SN-38. Although human lung cancer cells expressed hCE in the cytoplasm, hCE expression levels in cancer cells were not correlated with their drug sensitivities. Although intracellular CPT-11 and SN-38 levels continuously increased within 60 min of CPT-11 exposure, SN-38 levels in cells exposed to SN-38 decreased. Cells with the ability to metabolize SN-38 to SN-38G were more resistant to extracellular SN-38 than cells lacking the ability. Of 25 squamous cell carcinomas, 15 were strongly positive for hCE and six were negative. Of 25 adenocarcinomas, four were strongly positive for hCE and 16 were positive, while five were negative. Thus, 70% of non-small cell lung cancers expressed hCE. From these results, we conclude that human lung cancer cells expressed the enzyme which can convert CPT-11 to SN-38 and that intracellular SN-38 converted from CPT-11 may act as a chemotherapeutic agent together with SN-38 absorbed from the outside and augment the dose intensity of SN-38. Therefore, to assess the effects of CPT-11 prior to chemotherapy, it is important to check if lung cancer cells express hCE.