The alpha-subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length alpha-fodrin, but not by the anti-N-terminal sequence (GMMPR) of the mu-calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of alpha-fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified mu- and m-calpain did not catalyze the formation of the 120 kDa fragment from purified alpha-fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of alpha-fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.