Human mesothelial cells obtained from benign effusions retain their proliferative capacity and grow uniformly either with a fibroblastic or epithelioid morphology in vitro. These cultures therefore provide a model for the process of mesothelial differentiation in vivo. To study this differentiation, we isolated differentially expressed genes obtained by suppression subtractive hybridization. Of the 9 genes found to be overexpressed in fibroblastic mesothelial cells, 3 are matrix-associated (integrin 5, collagen binding protein 2, human cartilage glycoprotein 39), while the others are associated with a proliferative cell type (14-3-3 , plexin B2, N33, and three genes encoding ribosomal elements). Seven of the 8 genes upregulated in the epithelioid phenotype are related rather to specialized functions, such as metabolism (aldose reductase, lecithin:cholesterol acyltransferase, ATPase 6), cytoskeletal composition (cytokeratins 7 and 8) and regulation of differentiation (granulin, annexin II). Immunohistochemistry with available antibodies to 6 of the differentially expressed gene products confirmed the differences also in pleural tissues, where submesothelial cells displayed the fibroblastic while surface cells the epithelioid markers. In summary, this approach revealed a pattern of genes coordinately regulated during mesothelial differentiation and suggests that mesothelium may regenerate also by recruiting cells from the submesothelial layer. Some of the gene products may also be useful markers for differentiation and activation in serosal tissues.