Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) in vitro. We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4.Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of α-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of ?-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.