Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with additional Klenow treatment and direct sequencing mutations in the p53 tumor suppressor gene were analyzed from 21 cases of human astrocytomas. Three cases had p53 gene mutations: two of them were glioblastomas showing a point mutation, one in exon 5 and the other in 6. The last one was a gemistocytic astrocytoma showing a point mutation in exon 5. The frequency of p53 gene mutations in the astrocytomas examined was 14.3% (3 out of 21). No SSCP alterations were observed in any of the p53 fragments amplified from WHO grade I pilocytic astrocytomas and WHO grade III anaplastic astrocytomas. Further examination by direct sequencing showed that two mutations of glioblastomas had single-base substitutions resulting in silent and missense mutations, whereas one of the gemistocytic astrocytomas had a double-base substitution resulting in a missense mutation. The present studies revealed that all mutations were located outside the hot spots and, interestingly, one of them disclosed a missense mutation in exon 5 at codon 166, which was first detected in a grade II astrocytoma (gemistocytic type). It is possible that the missense mutation at this codon may be associated with special risk factors for the development of astrocytic tumors in Thai patients.