Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.