In chloroplasts isolated from susceptible and atrazine-resistant Amaranthus retroflexus, the inhibition of photosynthetic electron transport by various classes of herbicides has been investigated. Resistance of mutant Amaranthus is not restricted to s-triazines but also extends to uracils, 1,2,4-triazine-5-ones, and ureas. For 1,2,4-triazin-5-ones and chloroplasts of both biotypes, a correlation between inhibition of photosynthetic electron transport and the partition coefficient could be established. In the case of phenolic herbicides only modestly decreased or even higher sensitivity of chloroplasts from the resistant biotype as compared to the susceptible one could be observed. These results are confirmed by binding of radioactively labeled herbicides to chloroplasts of both plants. Specific binding of atrazine or metribuzin to resistant chloroplasts is completely abolished, and that of diuron or phenisopham diminished as compared to susceptible chloroplasts. In contrast, binding of phenolic herbicides generally is enhanced in resistant chloroplasts. Photoaffinity labeling of thylakoids from both biotypes by 2-azido-4-nitro-6-[2′,3′- 3H]isobutylphenol yields almost identical labeling patterns. These results are consistent with a recently proposed model (W. Oettmeier, K. Masson, and U. Johanningmeier, Biochim. Biophys. Acta 679, 376 (1982) of two different herbicide binding proteins at the reducing side of photosystem II: a 32- to 34-kdalton protein responsible for binding of triazines, triazinones, ureas, and related herbicides and a photosystem II reaction center protein for binding of phenolic herbicides.