The spontaneous emission of chemiluminescence by Trypanosoma cruzi epimastigotes was 133 +/- 5 counts s-1 (mg protein)-1. The measured intracellular steady state concentration of hydrogen peroxide in the same cells was 1.5 +/- 0.5 microM. These two values are about 12- and 15-times higher than the corresponding ones for isolated rat hepatocytes. The intracellular steady state concentrations of superoxide radical and hydrogen peroxide were apparently increased by inhibiting superoxide dismutase (with diethyldithiocarbamate or KCN addition) and by the addition of two different trypanocidal agents (beta-lapachone and nifurtimox) capable of intracellular redox cycling and in each case an increased chemiluminescence was observed. Depletion of intracellular reduced non-protein SH groups by 80% increased 3-fold the chemiluminescence of T. cruzi cells. It is apparent that both an increase in the intracellular steady state concentration of superoxide anion or hydrogen peroxide and a decrease in the level of reduced SH groups lead to an increase in the level of peroxy radicals which are the precursor species for light emission.