Two splice variants are derived from the BCL-x gene, proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L), via alternative 5' splice site selection. In previous studies, our laboratory identified an RNA cis-element within exon 2 of Bcl-x pre-mRNA that is a ceramide responsive termed CRCE 1. In this study, mass spectrometric analysis identified the splicing factor SAP155, as an RNA trans-acting factor binding to the purine-rich CRCE 1. The interaction of SAP155 with CRCE 1 was confirmed by the addition of an anti-SAP155 antibody (Ab) to EMSA decreasing the mobility of a protein:CRCE 1 complex (SuperShift). Furthermore, the down-regulation of SAP155 in A549 cells by RNA interference (RNAi) technology resulted in the loss of a 155 kDa protein complexed with CRCE 1. Moreover, this down-regulation of SAP155 induced an increase in the Bcl-x(s) with a concomitant decrease in the Bcl-x(L) splice variants and immunoreactive protein levels, thereby decreasing the Bcl-x(L)/Bcl-x(s) ratio. Specific down-regulation of SAP155 also inhibited the ability of exogenous ceramide treatment to further induce the activation of the Bcl-x(s) 5' splice site. Additionally, the specific down-regulation of SAP155 sensitized cells to undergo apoptosis in response to daunorubicin in a manner similar to ceramide. Therefore, we have identified SAP155 as an RNA trans-acting factor that binds to CRCE 1, functions to regulate the alternative 5' splice site selection of Bcl-x pre-mRNA, and is required for ceramide to induce the activation of the Bcl-x(s) 5' splice site. Furthermore, we have demonstrated that activation of the Bcl-x(s) 5' splice site can increase the effectiveness of chemotherapeutic drug treatment, thus establishing a role for the alternative splicing mechanism of Bcl-x in chemotherapeutic sensitivity.