When human cells incur DNA damage, two fundamental responses can follow, cell cycle arrest or apoptosis. Human RAD9 (hRAD9) and p53 function in both processes, but the mechanistic relationship between their activities is unknown. p53 mediates checkpoint control at G(1) by transcriptional regulation of p21. In this report, we show that hRAD9, like p53, can also regulate p21 at the transcriptional level. We demonstrate that overexpression of hRAD9 leads to increased p21 RNA and encoded protein levels. The promoter region of p21 fused to a luciferase reporter can be transactivated by either hRAD9 or p53, indicating that hRAD9 regulates the p21 promoter for transcriptional control of expression. Using an electrophoretic mobility-shift assay, we show that hRAD9 specifically binds to a p53-consensus DNA-binding sequence in the p21 promoter. Microarray screening coupled with Northern analysis reveals that hRAD9 regulates the abundance of other messages in addition to p21. Our data reveal a previously undescribed mechanism for regulation of p21 and demonstrate that hRAD9 can control gene transcription. We suggest that hRAD9 and p53 co-regulate p21 to direct cell cycle progression by similar molecular mechanisms. Furthermore, hRAD9 might regulate other cellular processes as well by modulating transcription of multiple down-stream target genes.