Extraneuronal accumulation of isoproterenol in atria and ventricle of perfused rat heart was investigated. Rat hearts were perfused with various concentrations of 3H-isoproterenol for 30 min in the absence and the presence of catechol-O-methyltransferase (COMT) inhibitor (tropolone). When COMT was intact, the accumulation of 3H-isoproterenol in both atria and ventricle after perfusion with low concentration of 3H-isoproterenol (0.01 to 1 mumol/l) was less than that of perfusing concentration; the tissue/medium ratio (T/M) of isoproterenol for artia was lower than that for ventricle. The T/M of isoproterenol after perfusion with 10 and 20 mumol/l of 3H-isoproterenol were 0.94 and 1.76 for atria and 3.25 and 2.95 for ventricle, respectively. When COMT was inhibited by tropolone, the T/M increased 6.3-9.0 folds for atria and 5.1-6.7 folds for ventricle after perfusion with 3H-isoproterenol (0.01 to 1 mumol/l). From these results, it was concluded that both atria and ventricle of the rat heart have an extraneuronal O-methylating system as reported in rat whole heart, and was suggested that there might be different capacities of extraneuronal uptake and COMT between them.