The hypothesis that human red cells contain activity of ribulose-1,5-diphosphate (Ru-1,5-P2) oxygenase, an enzyme that catalyzes the synthesis of phosphoglycolate, was examined. The putative Ru-1,5-P2 oxygenase was partially purified from human red cells using DE-52 chromatography and (NH4)2SO4 fractionation by monitoring Ru-1,5-P2-dependent 3-phosphoglycerate formation. The synthesis of [32P]phosphoglycolate from [1-32P]Ru-1,5-P2 was attempted in the presence of the partially purified preparation of the provisional Ru-1,5-P2 oxygenase. There was no formation of radioactive phosphoglycolate even under 100% oxygen gas, indicating the absence of this enzyme activity in human red cells. Together with our previous report that glycolate kinase in human red cells is not responsible for the synthesis of phosphoglycolate in vivo, these studies raise the questions whether there is actually phosphoglycolate in red cells as well as whether novel pathways for its synthesis exist.