Our study concerned the findings that rat and rabbit heart transplants do not survive after six hours. They become dark, hard and fail to contract within 2 min after reperfusion and never regain their function. We tested the supplementation of solutions for heart transplant preservation with tetrahydrobiopterin (H4B) and L-arginine (L-ARG) to maintain the oxidative and reductive domains of the endocardial NO synthase. We decided to study the excised rabbit hearts preserved in Hank’s balanced salt solution (HBSS) at 0 °C supplemented with different concentrations of H4B (0, 1, 5, 10 or 100 /¿M). At desired time intervals, successive pieces stored in the above solutions were warmed to rabbit body temperature in 4 ml of HBSS and maximally agonized by direct application of 20 l of 200 M bradykinin (or other agonist) onto the exposed endocardium. Nitric oxide bursts were monitored with a porphyrinic NO sensor lying on the exposed endocardium. Our goal was to find the lowest H4B concentration which would maximally agonize NO * and prolong the time of heart preservation to more than 6 hours. Ten /iM are a minimum H4B concentration which achieves maximum prolongation of heart preservation time up to 90 hours. This effect was based upon maximal potentiation of NO* release and minimizing of superoxide production.