Phosphoribulokinase from the nonsulfur purple bacterium Rhodopseudomonas acidophila has been purified to apparent homogeneity, using affinity chromatography on Cibacron Blue-agarose and AMP-agarose. The relative molar mass of the enzyme was determined by sucrose density gradient centrifugation to be Mr=248,000 with a sedimentation coefficient of s20,w=10.9 S. Dodecyl sulfate polyacrylamide gel electrophoresis revealed that the enzyme consists of identical size subunits of Mr=32,000, suggesting an octameric structure of the holoenzyme. The enzyme cross-reacted with heterologous antibodies raised against phosphoribulokinase from the hydrogen bacterium Alcaligenes eutrophus. The pH optimum of the enzyme was shifted from 8.4 in the absence of the activator NADH to 7.6 in the presence of the effector. Mg2+ ions were the most effective divalent cations required for activity. Specificity of the enzyme for the sugar phosphate substrate ribulose 5-phosphate was high whereas a variety of nucleoside triphosphates besides ATP could serve as phosphate donors. NADH was a strong activator of the enzyme (Ka=0.05 mM) that primarily affected the maximal reaction velocity in a pH-dependent manner. The only other effector identified was phosphoenolpyruvate. It moderately inhibited the enzyme (I0.5=0.32 mM).