SUMMARY The effectiveness of toxic oxygen metabolites in killing the yeast form of Paracoccidioides brasiliensis (the form that occurs in host tissues) was studied with a fluorescence method in vitro. The two isolates studied were similar in susceptibility and H2O2 alone was lethal with an LD50 of 15–25 mm. The addition of halide (5 × 10−4 M) augmented the lethality of H2O2 and in that setting H2O2 was c. 90% lethal at 5 × 10−5 M. Killing was most effective in the presence of peroxidase, when only 5 × 10−6 M H2O2 (a concentration attainable in vivo by phagocytes) was required for a 95% kill. Kinetic studies revealed that toxic concentrations of H2O2 alone or of the H2O2−halide-peroxidase (PPH) system produced significant killing in 1 min; killing was maximal in 15 min. The PPH system was the more rapid in action. The dependence of the PPH killing system on H2O2 was demonstrated by an absence of killing in the presence of catalase. The susceptibility of P. brasiliensis to H2O2 and the PPH system appeared different in some respects from that noted for other dimorphic fungal pathogens. These studies suggest that toxic oxygen metabolites are important in host defence against P. brasiliensis.