AbstractCell‐free preparations ofChlorella pyrenoidosaChick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared.The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose‐6‐phosphate, 6‐phosphogluconate, and fructose‐1‐6‐diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose‐diphosphate‐aldolase, NAD‐linked 3‐phosphoglyceraldchyde dehydrogenase, glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, and a NADP‐linked 3‐phosphoglyceraldchyde dehydrogenase.In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose‐6‐phosphate and 6‐phosphogluconate in relation to the reduction of NAD by 3‐phosphoglyceraldehyde was made in light‐ and dark‐grown cells. The rate of reduction of NADP appeared to be lowered in the light‐grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophi