Hypoxia is an important stimulus for blood vessel formation in many tissues and tumors. Identification of genes that are regulated by oxygen may therefore provide novel insights in the molecular mechanisms underlying this process. Recent findings suggest that the product of the Wilms' tumor gene, Wt1, whose expression is increased in hypoxia, has a role in neovascularisation of the ischemic heart. Here we demonstrate, that the integral membrane protein 2A (Itm2a), which has been implicated in myogenic cell differentiation, is up-regulated in response to hypoxia and Wt1. Itm2a mRNA levels were increased 2-fold in the hearts of mice following exposure to 8% O₂ for 6 h (students t-test, P<0.01, n=5). Incubation of the myoblast cell line C2C12 at 1% O₂ for 24 hours caused an 8-fold increase of Itm2a mRNA compared to cells at 21% O₂. A reporter construct carrying approximately 3 kb of the Itm2a promoter was stimulated 2.3-fold in transfected C2C12 cells grown at 1% O₂vs. 21% O₂ (P<0.05, n=3). Likewise, co-transfection of a Wt1 expression construct significantly enhanced Itm2a promoter activity in C2C12 cells. Endogenous Itm2a mRNA levels were reduced approximately 9-fold upon silencing of Wt1 by RNA interference in mesonephros-derived M15 cells. Immunohistochemical staining revealed co-expression of Wt1 and Itm2a in the epicardium of E11.5 mouse embryos. Itm2a transcripts were reduced 4-fold in Wt1-deficient embryos compared to their normal littermates (students t-test, P<0.05, n=5). In conclusion, stimulation of Itm2a expression in response to hypoxia and Wt1 may provide a novel regulatory mechanism in the heart.