Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Muell. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000+_ 10,000; a single band at 57,000 +_ 3,000 was detected after sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 +_ 30 mM for adenosine triphosphate and 40 +_ 2 mM for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 mM) and phosphoribosylpyrophosphate (inhibitor constant = 30mM) were inhibitors. PRS responded allosterically (Hills coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10mM). These results are set in the physiological context of laticifers.