It is now widely accepted that alternative splicing is a mechanism that is responsible for generating protein complexity at low genetic cost. However, little is known about molecular mechanisms that govern alternative splicing of key apoptotic regulators. Here we investigate the effect of pro-apoptotic stimuli on alternative splicing of Fas mRNA by means of reverse transcription-polymerase chain reaction (RT-PCR). Exposure of U937 cells to etoposide, staurosporine, pacritaxel, or cyclohexamide promoted the appearance of the splice variant, which retained the 152-base-pair intron 5. Pretreatment with calyculin A, an inhibitor of protein phosphatase-1 (PP-1) as well as fumonisin B1, an inhibitor of ceramide synthase, prevented etoposide-induced alternative splicing of Fas mRNA. Our data demonstrate that cross-talk between RNA splicing and signaling pathways through endogenous ceramide synthesis and subsequent phosphatase activation is a mechanism that modifies Fas gene expression at the posttranscriptional level.