The aim of this work was to investigate in the avascular heart of the frog Rana esculenta the influence of nitric oxide (NO) on ventricular systolic and diastolic functions by using a novel image analysis technique. The external volume variations of the whole ventricle were monitored during the heart cycle by video acquisition(visible light) and analysed by an appropriately developed software with a specific formula for irregular convex solids. The system, which measures the rate of volume changes and the ejection fraction, directly determined the volumetric behaviour of the working frog heart after stimulation or inhibition of NOS-NOcGMP pathway. End-diastolic volume (EDVext), end-systolic volume (ESVext), contraction and relaxation velocities (dV/dtsys and dV/dtdia, respectively), stroke volume (SV) and ejection fraction (EF), were measured before and after perfusion with NOS substrate (L-arginine), NO donor (SIN-1), cGMP analogue (8-Br-cGMP),NOS inhibitors (NG-monomethyl-L-arginine, L-NMMA; L-N(5)-(1-iminoethyl)-ornithine, L-NIO; 7-Nitroindazole,7-NI) and guanylyl cyclase inhibitor (ODQ). The results showed that NO reduces ventricular systolicfunction improving diastolic filling, while NOS inhibition increases contractility impairing ventricular filling capacity. The presence of activated eNOS (p-eNOS) was morphologically documented, further supporting that the mechanical activity of the ventricular pump in frog is influenced by a tonic release of NOS-generated NO.