Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.5 mg/ml) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 microM), provoked an accumulation of endogenous adenosine at a rate of 2.3 +/- 1.0 amol/cell per minute. A similar accumulation was observed with both unstimulated and stimulated PMNs after the addition of deoxycoformycin (dCF, 1-100 microM), an inhibitor of adenosine deaminase. Exogenous adenosine (10 microM) was deaminated at a rate of 9.8 +/- 3.7 amol/cell per minute in control or zymosan or fMLP-stimulated PMN suspensions. This deamination was nearly completely suppressed when the PMNs had been stimulated with PMA. In contrast, the activity of adenosine deaminase in PMN lysates (231 +/- 72 amol/cell per minute) was not modified by PMA stimulation. alpha, beta-Methyleneadenosine 5'-diphosphate (AMPCP, 2.5 mM), an inhibitor of membranous ecto-5'-nucleotidase, profoundly inhibited endogenous adenosine accumulation under all conditions. PMA stimulation also provoked an inactivation of extracellular adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in PMN suspensions. We concluded that PMNs, even when not stimulated, continuously produce adenosine by dephosphorylation of extracellularly released adenylates; and that stimulation of PMNs by PMA causes adenosine accumulation owing to the inactivation of adenosine deaminase released by broken cells.