RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor ␤1 (TGF-␤1). It is known that TGF-␤1 induces cell growth arrest by activating CDKN1A (p21 WAF1/Cip1 ), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-␤-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-␤-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.The RUNX family of transcription factors plays pivotal roles in normal development and neoplasias. In mammals, the RUNX family genes consist of RUNX1/AML1, RUNX2, and RUNX3 (see reference 37 for the nomenclature). All RUNX family members share the central Runt domain, which is well conserved and recognizes a specific DNA sequence, but each has relatively divergent N-and C-terminal regions. RUNX1 and RUNX2 are required for hematopoiesis and osteogenesis, respectively, and are genetically altered in leukemia and bone disease (15,20,26,27,29). In contrast, RUNX3 is involved in neurogenesis (10, 17) and thymopoiesis (36, 41) and functions as a tumor suppressor of gastric cancer (7,18). About 60% of primary gastric cancer specimens do not express RUNX3 due to a combination of hemizygous deletion and DNA hypermethylation of the RUNX3 promoter region (7,13,18,28).