Transforming growth factor β (TGF-β) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-β-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-β administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, αII-spectrin (α-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-β-induced apoptosis and αII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-β, nor was its substrate, poly(ADP-ribose) polymerase. These results identify αII-spectrin as a novel substrate that is cleaved during TGF-β-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of αII-spectrin during apoptosis and suggests that TGF-β induces apoptosis and αII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.