Cyclin-dependent kinase inhibitor p16(INK4a) is implicated in replicative senescence, cell immortalization, and tumor generation. However, the mechanism regulating its overexpression in senescent cells is unknown. We used the enhanced green fluorescent protein reporter system to scan regulatory elements in the upstream region of p16(INK4a). The results of 5'-deletion studies indicated that the transcription regulatory elements contributing to overexpression of p16(INK4a) in senescent cells were located in the region of the p16(INK4a) promoter from -622 to -280 bp. According to the results of in vitro DNase I footprinting, EMSA, and Southwestern blotting, we found a novel negative regulatory element, the INK4a transcription silence element (ITSE), at -491 to -485 bp of the p16(INK4a) promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16(INK4a) by interacting with the ITSE. The activity of the p16(INK4a) promoter increased significantly in young cells when the ITSE was deleted. The GC-rich region of the p16(INK4a) promoter from -466 to -451 was a positive transcription regulatory element. Deletion of this region showed 91.4% loss of p16(INK4a) promoter activity in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably.