Evidence is presented that the antitumor agent helenalin, a sesquiterpene lactone, suppresses anaerobic glycolytic enzymes of tumor cells at a number of sites and not exclusively at glycogen synthetase and phosphofructokinase, previously proposed sites for inhibition by alpha-methylene-gamma-lactones. Of the enzymes tested, the sulfhydryl-containing enzyme hexokinase was inhibited the maximum, i.e., 83%, by helenalin treatment, whereas phosphofructokinase and glycogen synthetase were suppressed approximately 45%. Another sulfhydryl-bearing enzyme, aldolase, was decreased approximately 43%. Phosphorylase a was inhibited 65%, glucose-6-phosphatase was inhibited 46%, and succinic dehydrogenase was inhibited 59% by helenalin treatment. Mitochondrial oxidative phosphorylation processes were also significantly depressed in the presence of helenalin in vitro with either succinate or alpha-ketoglutarate as substrates. Thus, a number of enzymes of anaerobic and aerobic carbohydrate metabolism of Ehrlich ascites cells appear to be inhibited by helenalin, which supposedly can alkylate functional groups, e.g., sulfhydryl groups of these enzymes, by a rapid Michael-type addition.