The development of heart failure is accompanied by an increase in TGFb expression and apoptosis induction. In cardiomyocytes of adult rat stimulation with TGFb results in restricted cardiac function and apoptosis. Previous studies suggest that NO is causally involved in TGFb induced apoptosis, since both molecules act via SMAD transcription factors and incubation of cardiomyocytes with TGFb increased nitric oxide (NO) release. This could be inhibited with the NO-synthase (NOS) inhibitor ETU. To discover the signalling pathway we performed real-time RT-PCR experiments and revealed that RNA expression of NOS isoforms was not increased. Instead phosphorylation of endothelial NOS was enhanced by TGFb. Incubation of cardiomyocytes with TGFb enlarged the number of apoptotic cells. This increase was blocked under NOS inhibition with the NO-synthase inhibitor ETU (1 mM), but not with iNOS specific inhibitor 1400W (100 nM) or nNOS specific inhibitor TFA (100 mM). TGFb induced apoptosis could be blocked by the inhibitor for soluble guanylate cyclase ODQ (10 mM) and the PKG inhibitor KT (1 mM). Apoptosis induction under TGFb was blocked by TGFb receptor I inhibitor SB431542 (1 mM), and reduced by inhibition of PI3-kinase with Ly (10 mM). In conclusion: this characterizes NO/cGMP pathway as mediator of TGFb induced apoptosis in cardiomyocytes and NO as a possible contributor to heart failure progression under TGFb.