The p16INK4A/MTS1 (p16) and p15INK4B/MTS2 (p15) genes map to 9p21 where genetic alterations have been frequently reported in various human tumors. Using the polymerase chain reaction (PCR), we investigated the loss of these genes on primary glioma samples and cultured glioma cells. All or any of three exons of the p16 gene were homozygously delted in 11 (35.5%) of 31 glioblastomas, none of 9 anaplastic astrocytomas and 5 astrocytomas, and in all 6 human glioma cell lines. Exon 2 of the p15 gene was homozygously deleted in 4 (12.9%) of 31 glioblastomas, but not in lower grade gliomas. It was homozygously deleted in 5 (83.3%) of 6 glioma cell lines. In 12 short-term cultures of cells derived from primary glioma samples, 5 (41.7%) and 2 (16.7%) glioblastoma-derived cells had homozygous deletion of all or any of the three exons of the p16 gene and exon 2 of the p15 gene, respectively. The deletion pattern of these genes in cultured cells was completely consistent with that seen in the primary tumors. Furthermore, two long-term cultures retained both genes that were identical to those in the original tumor tissues. Our results indicate that loss of the p16 and p15 genes may be involved in tumor progression in human gliomas, especially in the development of glioblastoma, that this loss may give growth advantage to the cells in culture, and that it is not the result of culture artifacts.