The ß-adrenoceptor antagonist propranolol has been successfully used to treat severe infantile hemangioma which is due to pathological blood vessel formation. However, the underlying mechanism is so far not known. In the present study the hypothesis that ß-receptor antagonists inhibit vasculogenesis of embryonic stem (ES) cells was investigated. In differentiating mouse ES cells CD31-positve endothelial cells as well as CD31-negative cells expressed the ß₁- and ß₂-adrenoceptor. Treatment of ES cells from day 3 to day 9 of cell culture with either the non-specific ß-adrenoceptor antagonist propranolol, the ß₁-adrenoceptor antagonist atenolol or the ß₂-adrenoceptor antagonist ICI-118,551 dose-dependent inhibited vasculogenesis of ES cells without exerting toxic effects. In contrast the percentage of spontaneously contracting embryoid bodies (EBs) was not affected, indicating specific effects on vasculogenesis. ß-adrenoceptors downregulated NO-generation in ES cells and dephosphorylated nitric oxide synthase 3 (NOS3), which is involved in vasculogenesis of ES cells. In contrast generation of reactive oxygen species (ROS) generation was not impaired. In summary our data suggest that ß-receptor antagonists inhibit vasculogenesis of ES cells by downregulating NO-generation.