Cell division is controlled by a series of positive and negative regulators which act at sequential points throughout the cell cycle. Disturbance of these checks could contribute to cancer by allowing excessive cell proliferation. The point in G1 at which cells irrevocably commit to DNA synthesis is controlled by protein complexes consisting of cyclin-dependent kinases (CDK4 or CDK6) and cyclins (D1, D2 or D3). These complexes are inhibited by low molecular weight proteins, such as p16INK4 (refs 1,2), p15INK4B (ref. 3) and p18 (ref. 4). Deletion or mutation of these CDK-inhibitors could lead to unchecked cell growth, suggesting that members of the p16INK4 family may be tumour suppressor genes. The recent detection of p16INK4 (MTS1) mutations in familial melanoma kindreds, many human tumour cell lines, and primary tumours is consistent with this idea. Previously, we described eight germline p16INK4 substitutions in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations predisposed carriers to melanoma, whereas two missense mutations had no phenotypic effect. We now describe biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. Only the melanoma-predisposing mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro. Our data provide a biochemical rationale for the hypothesis that carriers of certain p16INK4 mutations are at increased risk of developing melanoma.