A bstract. A supernatant fraction, free of plstids, was prepared by centrifugation from Euglena gracilis and used to ascertain whether or not the inhibition of carbon dioxide fixation by oxygen, known as the Warburg effect, is entirely independent of the light-driven phase of photosyn-thesis. This fraction exhibited in the dark the main features of the Werburg effect; namely, an inverse relationship between the degree of inhibition by oxygen and bicarbonate concentration, reversibility of the inhibition when the oxygen partial pressure is lowered and an increase in the proportion of 2-carbon compounds. It is proposed, therefore, that the inhibition by oxygen is manifest in the photosynthetic carbon reduction cycle and is independent of photosynthetic electron transport and phosphorylation.The inhibition of CO2 fixation by 02, in isolated spinach chloroplasts has recently been described in detail (3,6). It was then suggested that this inhibition, termed the Warburg effect, was primarily related to some aspect of the carbon reduction cycle rather than depression of activity of one of the light-driven reactions (photophosphorylation or photoreduction of NADP).In our experiments, we have used the supernatant fraction from Euglena gracilis from which plastids had been removed by centrifugation to obtain data consistent with this proposal. When provided with ribose-5-phosphate, ATP and NADPH, this preparation fixed 14CO2 in the dark at reasonable rates and in a linear fashion. Using this system, it therefore seemed probable that any contribution by the light driven phase of photosynthesis towards the mechanism of the Warburg effect could be detected.Three important characteristics of the Warburg effect are: an inverse relationsh-ip between oxygen sensitivity and bicarbonate concentration; a rapid reversal of this inhibition by reduction of the oxygen partial pressure and an increase in the synthesis of 2-carbon compounds !(glycolate in isolated chloroplasts and glycolate together with glycine in whole algal cells).