The p16(INK4a) protein is considered to regulate the cell cycle progression mainly by inhibiting cyclin-dependent kinases (CDKs) 4 and 6 activity and leading to an arrest in G(0)/G(1). Here, we report that ectopic expression of p16(INK4a) in three p16-/pRb(Wt)/p53(Wt) human cancer cell lines MCF7, U2OS and U87 induces S-phase lengthening along with G(1) accumulation. S-phase lengthening is suggested by the discrepancy between the unchanged or even increased percentage of cells in S phase found by flow cytometry DNA content analysis and the drop of BrdU labelling, and demonstrated by IdU/BrdU double labelling. p16(INK4a) induces a profound decrease in the CDK4/6-mediated pRb phosphorylation on Ser-807/811, a downregulation of CDK2 and CDK1 protein expression independently of G(1) accumulation, and a decrease in Thr/Pro phosphorylation in part carried out by CDKs. In MCF7 cells, overexpression of the p16 G101W mutant, which is unable to inhibit CDK4/6 kinase activity and shows a modified subcellular localization, does not provoke the S-phase lengthening and the inhibition of Ser807/811-pRB and of Thr/Pro phosphorylation as wild-type p16(INK4a) does. Our results demonstrate that p16(INK4a) induces a S-phase lengthening independently of cellular origin. The CDK4/6 kinase activity inhibition together with the reduced expression of CDK2 and CDK1 acting downstream of G(1) phase may prevent cells from any possible kinasic compensatory mechanisms, and thus lead to a cell cycle progression inhibition.