Human astrocytomas frequently have allelic losses of chromosome 9p, suggesting the presence of a 9p astrocytoma tumor suppressor gene. The MTS1 (or CDKN2) gene on chromosome 9p encodes a cell-cycle regulator and is deleted in approximately 80% of astrocytoma cell lines. To determine whether MTS1 is the tumor suppressor gene involved in human astrocytoma formation in vivo, we have analyzed chromosome 9p allelic loss and the MTS1 gene in 30 primary astrocytomas. Deletion mapping demonstrated 15 cases with allelic loss of chromosome 9p, with all losses either flanking or involving the MTS1 gene. Direct analysis of the MTS1 gene, however, revealed only a single missense mutation in a high-grade tumor that had lost the second allele. The low frequency of MTS1 mutations in primary astrocytomas with allelic 9p loss suggests that MTS1 may be more important for in vitro than in vivo astrocytoma growth, and that another 9p tumor suppressor gene may be involved in astrocytoma formation in vivo. Analysis of the MTS1 gene also demonstrated two intragenic polymorphisms, one in exon 2 and one in the 3' untranslated region, that can be used to assay allelic loss directly at MTS1.