Bladder instillation with Bacillus Calmétte-Guerin (BCG) is an established treatmentmodality for superficial high-risk urinary bladder-cancer and carcinoma in situ (CIS), but theanti-tumor mechanisms following BCG-instillations remain largely unknown. In this study weexamined the effects of BCG-treatment on SK1 protein expression in the murine bladdercancer cell line MBT2. To simulate in vivo BCG-instillations, where the immune systemplays a vital role for successful BCG-treatment, we also stimulated MBT2 cells withsupernatant from BCG-treated (SupBCG) or un-treated Raw 264.7 macrophages. BCGtreatment as well as SupBCG treatment induced SK1 protein expression. Treatment with thesphingosine-kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) induced reduced viability in a dose dependent manner. However, combined treatment withBCG diminished this viability reduction suggesting a protective effect of BCG, but not ofSup-BCG, from SK1-II induced toxicity. Apoptosis was detected, as PARP-cleavage, in cellstreated with SupBCG whereas BCG-only or supernatant from untreated macrophages did notinduce apoptosis. A substantial increase in ceramides C18, C18:1 and C20:1 was observedafter BCG-treatment. These ceramide subspecies, as well as ceramides C14, C16, C20, C22and C22:1, were also induced by 20 or 30M of SK1-II, but the induction was abrogated byco-treatement with BCG. Following treatment with Sup-BCG, the levels of all the abovementioned ceramide subspecies were increased. Levels of ceramides C14, C16, C18, C18:1,C20, C20:1, C22 and C22:1 were more than two times higher in response to combinedtreatment with Sup-BCG and 10M of SK1-II as compared to treatment with Sup-BCG orSK1-II alone. Taken together these data suggest the sphingolipid metabolism as an importantpathway in the response to BCG-therapy.