The effect of aureomycin on photosynthesis was investigated. This antibiotic which has been reported to stimulate photosynthesis at very low concentrations is an effective inhibitor at higher concentrations. In mesophyll protoplasts and isolated chloroplasts from spinach, 50% inhibition of CO2 reduction required about 20 μᴍ aureomycin. The reduction of 3-phosphoglycerate and of oxaloacetate by intact chloroplasts was also inhibited, but not that of nitrite and methylviologen which was actually stimulated. NADP reduction by broken chloroplasts and methylviologen- dependent photophosphorylation were also sensitive to aureomycin. The electrochromic shift at 518 nm which indicates formation of a light-dependent membrane potential was suppressed in the presence of 200 μᴍ aureomycin and the transthylakoid proton gradient was decreased. The data confirm reports that aureomycin has uncoupling properties, and they indicate that it also acts as an inhibitor of ferredoxin/NADP reductase.