Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.