Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non-erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during apoptosis may have implications for the membrane blebbing seen during this process.