The p16INK4 gene is a candidate tumour-suppressor gene which maps to the genomic locus 9p21, and mutations of this gene are associated with melanoma and other cancers. Biochemical studies suggest that p16INK4 mediates its effects by specifically inhibiting the G1 cyclin-dependent kinases CDK4 and CDK6, thereby regulating the progression through G1 into S phase of the cell cycle. To evaluate the functional effects of mutations in p16INK4 which have been observed in primary cancers and cancer cell lines, we constructed a series of deletion mutants comprising amino acid regions 9-72, 9-131, 73-131 and 73-156; a mis-sense mutation identified in melanoma (Arg87Pro); and the polymorphism Ala48Thr and investigated their ability to inhibit cyclin D1/CDK4 kinase activity in vitro. Removal of 25 amino acids from the carboxy terminus of p16INIK4 (9-131) had little impact on its inhibitory activity. In contrast, deletion of the 65 N-terminal amino acids comprising the first and second ankyrin repeats of p16INK4 (73-131) abolished its inhibitory activity. The carboxy (73-156) and amino termial (9-72) fragments of p16INK4 also failed to inhibit cyclin D1/CDK4 activity. These results indicate that the core region (73-131) as well as amino acids N-terminal of this sequence are important, whereas sequences C-terminal of amino acid 131 are less important for the inhibitory activity of this molecule. The melanoma-associated Arg87Pro mutation resulted in loss of inhibitory activity, whereas the Ala148Thr polymorphic variant was as effective as the alanine variant of p16INK4 in inhibiting D1/CDK4 kinase activity. Binding assays revealed that inhibition was invariably associated with p16INK4 binding to CDK4. Hence, our studies indicate that minor perturbations in p16INK4 primary structure can lead to loss of its inhibitory activity, possibly contributing to oncogenesis in numerous cell types.