The p16 protein plays a key role in cell cycle control by preventing CDK4 from inactivating the retinoblastoma protein (pRb). The corresponding tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicated in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 methods of detecting p16 deletion. Thirty diffuse gliomas were analyzed for p16 gene dosage. Dual color fluorescence in situ hybridization (FISH) was performed on cytologic preparations using paired centromeric (CEN) and locus-specific probes for CEN9/p16, CEN8/RB, and CEN12/CDK4. Quantitative PCR was performed using primers for p16, MTAP, and reference genes. Eleven cases were also studied using comparative genomic hybridization (CGH). Abnormalities of the p16-CDK4-RB pathway were identified in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis further revealed tetraploidy/aneuploidy in 14 (47%), RB deletion in 11 (37%) and CDK4 amplification in 1 (3.3%). There were 94% and 100% concordance rates between CGH and FISH or PCR, respectively. Quantitative PCR was noninformative in 4 cases. Although FISH and quantitative PCR are both reliable techniques, each has limitations. PCR is likely to miss p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysis may miss small deletions. Replacement of the latter with a cosmid probe may improve the sensitivity of FISH in future experiments.