We have investigated the kinetic properties of the extracellular reaction sequence ATP----ADP----AMP----adenosine catalyzed by ectonucleotidases at the surface of adult rat cardiac myocytes. Analysis of progress of reaction curves indicates that depletion of substrate at cell surfaces dominates the regulation of the rate of hydrolysis of ATP or of ADP when it is the initial substrate. Preferential delivery of intermediate products to be substrates at cell surfaces makes a significant contribution to the regulation of adenosine production from ATP or ADP. Preferential delivery has more impact on the delivery of ADP from adenosinetriphosphatase (ATPase) to adenosinediphosphatase (ADPase) than on delivery of AMP from ADPase to 5'-nucleotidase. At high initial ATP concentrations, feed-forward inhibition of AMP hydrolysis also modulates the rate of adenosine production. Taken together, the properties of the ectonucleotidases on the myocyte provide a milieu at the cell surface that tends to be poor in nucleotides, especially ATP and ADP (P2 purinoceptor agonists), and rich in adenosine (a P1 purinoceptor agonist) during periods of supply of extracellular nucleotides.