Our study examines the effect of apoptosis on prothymosin ␣, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin ␣ levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin ␣. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin ␣. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin ␣. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin ␣ was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin ␣ is a significant event in apoptosis.