Expression of full-length p16(INK4a) blocks alpha(v)beta(3) integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G(1)-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alpha(v)beta(3)-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G(1). The alpha(v)beta(3) heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts, Treatment with phorbol 12-myristate 13-acetate allows alpha(v)beta(3) to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides, The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading, These results demonstrate a novel G(1) CDK-associated integrin regulatory pathway that acts upstream of alpha(v)beta(3)-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.