In perfused isolated guinea pig hearts reactive hyperemia (RH) was induced by occlusion of coronary flow for periods ranging from 1-60 s. RH was hampered by 100-60% in the presence of an inhibitor of NO synthase, NG-nitro-L-arginine (100 microM) and, to a lesser extent (up to 35%), by an antagonist of adenosine receptors, 8-phenyltheophylline (10 microM). An inhibitor of PGH synthase, indomethacin (5 microM), did not affect RH. During RH the heart generated prostacyclin, nitric oxide, and adenosine as indicated by the appearance of 6-keto-PGF1 alpha, cyclic GMP, urate, inosine, hypoxanthine and xanthine in the perfusate. Out of these factors only NO and adenosine were responsible for RH. NO was responsible for RH which was evoked by short-term (1-10s) coronary occlusion, whereas concurrent efforts of NO and adenosine were required to maintain RH that followed longer (20-60s) periods of interruption of coronary inflow. Thus, in the investigated system nitric oxide and adenosine but not prostacyclin can be considered as the mediators of myocardial reactive hyperemia.