This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix (14)CO(2) when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial (14)CO(2) fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of (14)CO(2) fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no (14)CO(2) fixation occurred when 3-phosphoglycerate was used. (14)CO(2) fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5'-phosphate. Low concentrations of O(2) (25-50 micromolar) stimulated (14)CO(2) fixation, but the activity decreased with increasing O(2) concentrations. The fixation of (14)CO(2) in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.