Apoptosis of cardiomyocytes plays an essential role in the development of post-ischemic heart failure. Within several pathways modulating the apoptosis in cardiomyocytes, cAMP signalling has been shown to play an essential role. Aside from the G-protein-responsive transmembrane adenylyl cyclase, ubiquitous expression of soluble adenylyl cyclase (sAC) has been demonstrated. However, the role of this cyclase in cardiomyocyte apoptosis was unknown. To investigate this subject, primary culture of rat ventricular cardiomyocytes was subjected to in vitro ischemia (3 h, glucose free anoxia at pH 6.4) and reperfusion (6 h, standard cell culture medium). Apoptosis was analysed by DNA-ladder, chromatin staining and caspase-3 cleavage. Although ischemia alone had no effect on apoptotic cell death, reperfusion within 6 hours led to significant rise of apoptotic cell number (18.4+2.3% vs. 6.1+1.8% in control). Suppression of the sAC activity during the ischemic phase with the selective inhibitor KH7 (20 mM, ChemDiv) decreased cellular cAMP content and prevented apoptosis in cardiomyocytes at the end of reperfusion (8.3+1.4%). In contrast, treatment with inactive analogue of KH7, i.e. KH7.14 (ChemDiv) had no effects on cAMP content and apoptosis. Analysis of the underlying mechanisms of the sAC-dependent apoptosis revealed activation of the mitochondrial (caspase-9 cleavage) and sarcoplasmatic reticulum (caspase-12 cleavage) apoptotic pathways. Both pathways were suppressed by inhibition of sAC with KH7. Finally, analysis of reactive oxygen species (ROS) with dichlorofluorescein demonstrated a burst of ROS production during 60 min reperfusion, which was completely suppressed by sAC inhibition. Taken together, these results suggest that sAC plays a significant role in the apoptosis of cardiomyocytes under ischemia-reperfusion by controlling ROS production and caspase-12/-9 cleavage.