Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.