The vast majority of glioblastomas have CDKN2A, CDK4, or RB gene alterations that perturb the p16-cdk4-pRb cell cycle regulatory cascade. To explore whether immunohistochemical methods provide an alternative means of assessing this pathway, we studied 25 glioblastomas using a combination of molecular genetic and immunohistochemical assays. Homozygous deletion of the CDKN2A gene was detected in 12 of 25 (48%) cases, CDK4 amplification in 4 of 25 (16%) tumors, and loss of heterozygosity at the RB gene in 8 of 22 (36%) informative cases. Five of 25 (20%) glioblastomas had diffuse p16 immunohistochemical positivity. Significantly, all of these had either CDK4 amplification or RB LOH, suggesting that p16 immunopositivity only occurs in those tumors with alterations of another component in the pathway. Nineteen (76%) cases were uniformly immunonegative for p16, and 12 (48%) had CDKN2A homozygous deletions, but the remaining 7 cases lacked CDKN2A deletions, mutations and promoter methylation. All glioblastomas stained diffusely for cdk4, irrespective of CDK4 gene amplification status. Extensive pRb staining was present in most cases that maintained both RB alleles, and absent in most cases with RB loss, but there were notable discrepancies. Thus, p16 and pRb immunohistochemistry cannot replace molecular genetic analysis of this critical regulatory cascade; instead, the combined results hint at complex regulation of this cell cycle checkpoint. From a practical point of view, although p16 immunonegativity does not necessarily indicate CDKN2A deletion, diffuse positive p16 immunostaining strongly suggests either CDK4 amplification or RB loss and excludes CDKN2A deletion.