The in vitro effects of H2O2 on platelet adenine nucleotide metabolism and on the ADP-induced platelet release reaction were examined. All studies were performed on human platelet-rich plasma (PRP) preincubated with (3H)-adenine. Within 3 min of incubation with PRP, H2O2 (100-500 micronM) caused an irreversible reduction in the (3H)-ATP level with a concomitant increase in (3H)-IMP and (3H)-inosine and hypoxanthine levels. The adenylate energy charge (AEC) initially decreased while the ATP level fell. The AEC, however, returned to levels slightly lower than the control during further incubation. No leakage of ATP and ADP to plasma was observed. The fall in the steady-state levels of (3H)-ATP increased with increase of the H2O2 concentration (decrease of 8.7-40% at H2O2 concentrations from 5 to 600 micronM). H2O2 pretreatment of PRP caused absence of ADP-induced biphasic aggregation, partial inhibition of the primary wave, and complete inhibition of release of platelet nonmetabolic ATP and ADP. Our in vitro findings support the view that part of the inhibitory effect of H2O2 may be related to the lowering of metabolic ATP levels in platelets.