Diffuse astrocytoma WHO grade II is a well-differentiated, slowly growing tumor that has an inherent tendency to progress to anaplastic astrocytoma (WHO grade III) and, eventually, to glioblastoma (WHO grade IV). Little is known about its molecular basis, except for p53 mutations that are found in >60% of cases. In a search for additional genetic alterations, we carried out gene expression profiling of 11 diffuse astrocytomas using cDNA expression arrays. Expression of six genes (TIMP3, c-myc, EGFR, DR-nm23, nm23-H4, and GDNPF) was detected in 64-100% of diffuse astrocytomas, but not in nontumorous brain tissue. Seven genes (AAD14, SPARC, LRP, PDGFR-alpha, 60S ribosomal protein L5, PTN, and hBAP) were found to be up-regulated more than 2-fold in 20-60% of cases, whereas 11 genes (IFI 9-27, protein kinase CLK, TDGF1, BIN1, GAB1, TYRO3, LDH-A, adducin 3, GUK1, CDC10, and KRT8) were down-regulated to less than 50% of normal levels in 64-100% of cases. Semiquantitative conventional reverse transcription-PCR was performed for 11 genes, 9 of which showed an expression profile similar to that obtained with cDNA expression arrays. Immunohistochemical staining for SPARC showed cytoplasmic immunoreactivity of neoplastic cells in all diffuse astrocytomas analyzed. These results indicate significant changes in gene expression in diffuse astrocytomas, but it remains to be shown which of these are causally related to the transformation of glial cells.