Under different pathophysiological conditions, for example after myocardial infarction, the growth factor TGFb is increased in hearts and contributes to cardiac remodelling. Since there is a diversity of sources of TGFb expression in heart we now analyzed if cardiac endothelial cells are a source of bioactive TGFb under hypoxic (Hx) and reoxygenated (Rx) conditions, and which signaling pathways are induced by TGFb. Microvascular endothelial cells were isolated from rat hearts and were exposed to hypoxia (Hx) for 2 hours and reoxygenated (Rx) up to 24 hours. The TGFb precursor protein expression was increased significantly after 1h and 24h Rx ((n=21, p<0.05). In order to analyse, if increase of TGFb precursor protein results in enhancement of bioactive TGFb, we analysed activation of SMAD transcription factors, as classical signaling molecules of TGFb. An increase in phosphorylation of SMAD2 to 130 ± 15% or 113 ± 4% (n=13, p<0.05) was found after 2h Hx/2h or 24h Rx. This effect was abolished by incubation of endothelial cells with TGFb type I receptor blocker SB431542 (1mM). Addition of supernatants from endothelial cells after 1h Rx to ventricular cardiomyocytes enhanced phosphorylation of SMAD2 also in cardiomyocytes within 1.5h to 112 ± 1% (n=4, p<0.05). This indicates that endothelial cells release active TGFb that can act on other cell types. Simultaneously with the induction of P-SMAD2 after 2 h Rx in endothelial cells the transcription factor GATA2 increases to 126 ± 10 % (n=11, p<0.05), whereas after 24 h Rx, enhancement of GATA2 was not present. This indicates a TGFb induced gene regulation by GATA2/SMAD2 interaction at early time points of reoxygenation, whereas after 24h Rx SMAD2 interacts probably with different binding partners. In conclusion, under hypoxic conditions endothelial cells release TGFb at early and late time points. This can induce SMAD2/GATA2 dependent processes of transcription in cardiomyocytes as well as in endothelial cells.