The pathway of production of AMP and adenosine in the myocardial interstitial fluid compartment was studied in the isolated perfused normoxic guinea pig heart by collecting the transmyocardial effluent (t.m.e.) with the method of De Deckere and Ten Hoor (1977). Besides adenosine and inosine, AMP was found in t.m.e. Infusion of α,β-methylene adenosine 5′-diphosphate (AOPCP), a specific inhibitor of the ecto 5′-nucleotidase, resulted in increases in t.m.e. AMP and inosine and a decrease in adenosine. Infusion of acetate producing a nearly twofold increase in myocardial AMP content did not increase the t.m.e. AMP even in the presence of AOPCP. In preparations made from 6-OH dopamine treated animals, the t.m.e. adenosine and inosine were reduced and AOPCP produced smaller increases in AMP and inosine, indicating that most if not all of the t.m.e. AMP originated from the sympathetic nerve terminals. Infusions of β,γ-imidoadenosine and β,γ-methylene adenosine 5′-triphosphate (AMPPNP and AMPPCP), non-hydrolysable analogs of ATP, resulted in dose-dependent increases in the t.m.e. AMP, which were much augmented in the presence of AOPCP. AMPPNP produced similar effects in 6-OH dopamine-treated preparations. As AMPPNP and AMPPCP are good substrates of ATP pyrophosphohydrolase, these findings indicate the presence of ATP pyrophosphohydrolase in the myocardial interstitial space.