Mutation of the p53 gene is found in about one third of astrocytic brain tumors, and expansion of tumor cell clones containing mutant p53 has been implicated in astrocytic tumor progression. However, admixture of normal cells in astrocytic tumor specimens limits the power of traditional studies of tumor cell clonality. To address this problem we have employed a yeast p53 functional assay that scores the content of mutant p53 alleles in tumors and cell lines quantitatively. We have analyzed 17 cases where matching tumor material and derived cell lines were available. The yeast assay gave > 20% red (i.e., mutant p53-containing) yeast colonies in 7 out of 17 cases. One case had no mutations in the primary tumor but gave 76% red colonies in a recurrence, clearly demonstrating tumor overgrowth by a mutant clone. During early passages of cultured tumor cells, mutant p53 content increased rapidly with passage due to outgrowth of mutant clones from a heterogeneous starting population. In addition, de novo p53 mutations appeared during culture in 2 cases. This indicates that there is stronger selective pressure for mutation during the establishment of cell lines in vitro than during tumor growth in vivo. Our results demonstrate the utility of the p53 functional assay for studies of clonality and support the hypothesis of clonal progression of brain tumors in vivo.