Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, two specific inhibitors of the b-c1 segment of the respiratory chain, affected the respiration of Trypanosoma cruzi epimastigote forms. The half-maximum inhibitory concentrations were about 0.05 and 4.0 micrograms/mg cells (dry wt.), respectively. The maximum effect of antimycin (about 80% inhibition of respiration) was at about 0.1 microgram antimycin/mg cells. Differential spectrophotometry of T. cruzi epimastigotes in the presence of antimycin, cyanide (or sulfide) and uncouplers, revealed the presence of functional cytochromes aa3, b and c558. In the stationary growth phase respiration by T. cruzi was completely inhibited by cyanide and effectively inhibited by sulfide, but in the exponential growth phase respiration was about 20% insensitive to 5 mM cyanide. Cyanide- and antimycin-insensitive respiration was completely inhibited by salicylhydroxamic acid (2 mM). Antimycin inhibited the operation of the tricarboxylic acids cycle in T. cruzi, as shown by the lesser production of 14CO2 and by the modification of 14C distribution in epimastigotes incubated with [1-14C]glucose, [2-14C]acetate or NaH14CO3. The inhibition of electron transport by antimycin increased the rate of the fumarate reductase reaction, an alternative electron pathway for the oxidation of reduced pyridine nucleotides. Addition of carbonyl cyanide 3-chlorophenylhydrazone to epimastigotes increased the rate of respiration and promoted the oxidation of reduced cytochrome b components, thus showing that these components are subject to respiratory (acceptor) control. Pentachlorophenol similarly affected the cytochrome b redox level but did not modify the rate of respiration. The uncouplers released N,N'-dicyclohexylcarbodiimide inhibition of respiration, and uncouplers and cyanide significantly decreased the ATP level in epimastigotes. The combined effects of the assay inhibitors on respiration, cytochrome b redox level, ATP content and energy charge confirmed the operation of oxidative phosphorylation in T. cruzi epimastigotes. Antimycin, uncouplers and N,N'-dicyclohexylcarbodiimide inhibited growth of T. cruzi, thus proving the essential role of oxidative phosphorylation for the parasite.