Despite the high response rates of small cell lung cancer (SCLC) to first-line cisplatin-based chemotherapies, most patients with SCLC will eventually experience disease progression. Accordingly, novel chemotherapeutic regimens are desired. This in vitro study was carried out in order to develop novel chemotherapeutic regimens containing 5-fluorouracil (5-FU) or oral fluoropyrimidine for SCLC. 5-FU was combined with other standard drugs for SCLC (cisplatin, etoposide, an active metabolite of irinotecan and amrubicin) in different schedules. The combination effects were analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and an isobologram method using H69 SCLC cells. Among the examined combinations, synergistic growth inhibition was observed only when H69 cells were treated with 7-ethyl-10-hydroxycamptothecin (SN-38; an active metabolite of irinotecan) followed by 5-FU. The findings of a flow cytometric analysis were consistent with the enhancement of apoptotic cell death by this sequential treatment. This synergism was observed in 4 out of 5 SCLC cell lines tested. The effects of 5-FU and SN-38 on thymidylate synthase (TS) protein expression, an important determinant of 5-FU sensitivity, were assessed by Western blot analysis in H69 cells. Treatment with SN-38 for 24 h suppressed TS protein expression and this low level of TS was maintained for at least 72 h. Pretreatment with SN-38 inhibited the 5-FU-induced increase of TS protein. The synergistic effect induced by the combination of SN-38 and 5-FU may be attributable to the SN-38-induced suppression of TS protein. Furthermore, uracil and 5-chloro-2,4-hydroxypyridine, which are clinically available dihydropyrimidine dehydrogenase inhibitors, enhanced 5-FU-induced growth inhibition. These observations provide evidence supporting the clinical applications of the combination chemotherapy using irinotecan and 5-FU or oral fluoropyrimidines against SCLC.