p27^Kip1, a cyclin-dependent kinase inhibitor, is recognized as a negative regulator of the cell cycle. In this paper, we report that overexpression of p27^Kip1 triggers apoptosis in several different human cancer cell lines. Using a recombinant adenoviral vector that expresses p27^Kip1 (Adp27), we found that overexpression of p27^Kip1 in MDA-MB-231 breast cancer cells induces apoptosis that was seen by a number of different techniques, including flow cytometry and in situ terminal deoxynucleotidyl transferase-mediated nick end labeling, flow cytometric assay for sub-G₁ population, and 4′,6-diamindino-2-phenylindole staining. Cleavage of poly-(ADP-ribose) polymerase and degradation of cyclin B1, events that are known to be associated with apoptosis, were also observed following overexpression of p27^Kip1. This is the first report indicating a role for p27^Kip1 in induction of apoptosis.