The development of methods of preparation of long wavelength ultraviolet light capable of activating ribulose bisphosphate carboxylase is reported. This preparation was obtained from tobacco (Nicotiana tabacum) leaves which had reached about one-half maximum leaf weight. It was prepared at low ionic strength by use of mixed anion and cation exchange resins and buffers containing dimethylsulfoxide. The preparation is greatly enriched in fraction I protein to the point of apparent homogeneity. When assayed in the presence of saturating ribulose bisphosphate and sodium bicarbonate, the rate of carbon fixation is a linear function of long wavelength ultraviolet irradiation in the range of 20,000 to 30,000 ergs per square centimeter per second. Glutathione (5 mm) inhibits light activation without affecting activity in the dark. Copper sulfate inhibits both light and dark activity, but is slightly less effective in the presence of ultraviolet light. Sucrose inhibition of carboxylation is only readily apparent in the absence of ultraviolet light. Ammonium sulfate precipitation followed by solubilization in buffers containing dimethylsulfoxide plus heat treatment promotes ultraviolet light activation.