The p16/CDKN2 gene has many features of a growth suppressor gene: it maps to 9p21, a frequent region of loss of heterozygozity in a variety of tumor types; it encodes an inhibitor of cyclin-dependent kinase 4; and its homozygous deletion is common in tumor-derived cell lines. However, the lower frequency of alteration of the gene in primary tumor tissue as compared to the cognate tumor cell lines has brought this interpretation into question. We have assessed the growth suppressive function of p16/CDKN2 by gene transfer. The introduction of full-length p16/CDKN2 cDNA caused marked growth suppression in p16/CDKN2-null human glioma cells, but was without significant effect in those cells with endogenous wild-type p16/CDKN2 alleles. These results provide functional evidence in support of the hypothesis that the p16/CDKN2 gene is a functional growth suppressor gene, at least in gliomas.