The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16 Ink4a. ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19 ARF. Little is known about the dependency of human p14 ARF function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14 ARF-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53+/+ p21 CIP1+/+ 14-3-3σ+/+), but not p53−/− counterparts, underwent G 1 and G 2 cell cycle arrest following infection with a p14 ARF-adenovirus. In p21 CIP1−/− cells, p14 ARF did not induce G 1 or G 2 arrest, while 14-3-3σ−/− counterparts were mainly arrested in G 1, pointing to essential roles of p21 CIP1 in G 1 and G 2 arrest and cooperative roles of p21 and 14-3-3σ in ARF-mediated G 2 arrest. Our data demonstrate a strict p53 and p21 CIP1 dependency of p14 ARF-induced cell cycle arrest in human cells.