The effects of hydrogen peroxide (H2O2) on the purine metabolism of human endothelial cells were investigated. An incubation with 0.01 mM H2O2 over 60 min led to an increase in the intracellular adenosine-5-triphosphate (ATP) and creatine phosphate (CP) levels by 51.3% and 18.2%, respectively. A 60 min incubation with 0.1 mM H2O2 showed no effect. The uptake and salvage of 14C-adenine (14C-AD) and 14C-adenosine (14C-ADO) was significantly (p < 0.005) increased using 0.01 mM H2O2. Only an increase of 14C-ADO incorporation was observed using 0.1 mM H2O2. A concentration of 0.01 mM H2O2 reduced 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP-S) activity by 60% and at the same time increased the activity of purine nucleoside phosphorylase, which converts inosine to hypoxanthine (PNP I), by 24%. Adenosine kinase (AK) activity was reduced by H2O2, whereas adenine phosphoribosyltransferase (APRT) activity was found to be elevated. In conclusion, the observed elevation of cellular ATP and CP levels could be partially caused by an increased purine salvage resulting from changes in purine enzyme activities.