Germline mutations of the CDKN2A gene are found in melanoma-prone families and individuals with multiple sporadic melanomas. The encoded protein, p16INK4A, comprises four ankyrin-type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16INK4a with its physiological targets, the cyclin-dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley-Liss, Inc.