Antimycin has been used to study the role of cyclic electron transport in isolated intact chloroplasts maintained under aerobic conditions. At all light intensities, antimycin inhibits CO2 fixation when assay conditions are optimal. When turnover of the Calvin cycle is inhibited, antimycin stimulates bicarbonate-dependent O2 evolution. Energy-dependent processes such as chlorophyll a and 9-aminoacridine fluorescence quenching, and light-scattering (apparent absorption) changes are inhibited by antimycin. The results suggest that cyclic electron transport contributes to photophosphorylation under aerobic conditions and is obligatory as a source of ATP during the most active periods of CO2 fixation in vivo. Cyclic electron transport can be stimulated either by inhibiting Photosystem II activity or increasing the turnover of Photosystem I relative to Photosystem II. These effects are interpreted in terms of the need for correct redox poising of carriers in the pathway in order to sustain maximum rates of cyclic electron flow. Binding studies indicate the presence of a high affinity antimycin binding site on chloroplast membranes. The stoichiometry and dissociation constant of the high affinity site are consistent with the idea that antimycin inhibits cyclic electron transport by binding to a b-type cytochrome in the thylakoid membrane.