In the present study we investigated the frequency of p16 gene exon 2 mutations in 35 malignant gliomas, using either direct sequencing of the PCR products or cloning into the pCRII vector and sequencing of the cloned PCR products. No mutations were detected during direct sequencing of the PCR products. However, after sequencing of individual clones, we found multiple mutations in 5 tumors involving codons 73(GCC to ACC, Ala to Thr), 76 (GCC to GTC, Ala to Val), 85(GCT to ACT, Ala to Thr), 98(CAC to TAC, His to Tyr), 102 (GCG to GTG, Ala to Val), 106 (GTG to ATG, Val to Met), 107 (CGC to TGC, Arg to Cys), 127 (GCA to GTA, Ala to Val), 128 (CGG to TGG, Arg to Trp) and 136 (GGC to GAC, Gly to Asp). Mutations were found only in glioblastomas and were either C to T or G to A transitions. Each mutation was detected in a small percentage of tumor cells (1.3-22%) using individual colony sequencing and southern hybridization with mutant oligonucleotides, consistent with the heterogenous cell population of glioblastomas. The presence of p16 gene mutations only in glioblastomas suggests that they are late events in glioma development.