Addition of beta-lapachone to the epimastigote (culture) form of Trypanosoma cruzi, suspended in saline, buffered-isotonic medium (pH 7.2), determined the appearance of large amounts of H2O2 in the suspension medium, as measured spectrophotometrically by formation of the H2O2 horse radish peroxidase complex. Under similar conditions, alpha-lapachone did not induce H2O2 formmation. Using NADH as electron donor, beta-lapachone (not alpha-lapachone) increased significantly the rate of H2O2 generation by epimastigote homogenates and the same occurred with NADPH, although in a reduced extent. Similar results were obtained with the isolated mitochondrial and microsomal fractions although with the latter NADPH was more effective than NADH as electron donor for beta-lapachone reduction and peroxide generation. The distribution of peroxide generation in epimastigote fractions would indicate that about 92% of the beta-lapachone dependent formation of peroxide occurred in the mitochondria, and 8% in the endoplasmic reticulum. The growth of epimastigotes was inhibited 95% by 1 microgram/ml beta-lapachone, a concentration that determined maximal rate of H2O2 production. Since H2O2 and other intermediates of oxygen reduction such as O2- (superoxide anion) and OH (hydroxyl radical) are lethal to cells and tissues, it is possible that the effect of beta-lapachone on T. cruzi proliferation in vitro was mediated by H2O2 and related free radicals.