Proteinase 3 (PR3), also called myeloblastin, is involved in the control of myeloid cell growth, but the underlying molecular mechanisms have not been elucidated. In U937/PR3, stably transfected with PRCRSV/PR3 to overexpress PR3, PMA-induced p21 expression was significantly decreased as compared with control U937, and this phenomenon was reversed in the presence of the serine proteinase inhibitor, pefabloc. Conversely, when PR3 was inactivated by small interfering RNA, p21 protein was increased, and PMA-induced monocytic differentiation was potentiated. Mass spectrometry analysis identified Ala45 as the primary cleavage site on p21, and the recombinant mutated p21A45R, generated by site-directed mutagenesis and expressed in Escherichia coli, was resistant to in vitro PR3 cleavage. The U937 cells were then stably transfected with either PRCRSV/p21 or PRCRSV/p21A45R, to ectopically express wild type p21 or PR3-resistant p21, respectively. In U937/p21A45R treated with PS-341, a selective proteasome inhibitor, a significant decrease in the S phase and a blockade in the G0-G1 phase of cell cycle were observed when compared with U937/p21 or control U937. This suggested that both PR3 and the proteasome are efficiently involved in the proteolytic regulation of p21 expression in myeloid cells. Moreover, PMA-induced p21 expression was more pronounced in U937/p21A45R compared with U937/p21 and was concomitant with the morphological features of early differentiation. Our data demonstrated that p21 is one specific target of PR3 and that PR3-mediated p21 cleavage prevents monocytic differentiation.