Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.Phosphoriboisomerase (EC 5.3.1.6) participates in the interconversion ofribose-5-phosphate and ribulose-5-phosphate in both the oxidative and reductive pentose phosphate pathways. We have now developed a rapid procedure for the isolation of the chloroplastic phosphoriboisomerase from pea (Pisum sativum) shoots. Although the enzyme has been purified from other sources (24) and spinach ( 18), this is the first time that the enzyme has been purified to apparent homogeneity from the chloroplasts of a higher plant.We have characterized the enzyme with respect to kinetic parameters and inhibitor constants, and we have determined the N-terminal sequence through 34 amino acids. Phase partitioning experiments indicate that the chloroplast phospho-'In memory of Professor Mordhay Avron.