Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose‐6‐phosphate isomerase, glyccrol‐3‐phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, β‐hydroxyacyl‐CoA dehydrogenase, 3‐ketoacid CoA‐transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and TA. Group OX enzymes increased 56‐193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated for a conversion of about half of the originally predominant fast glycolytic fibers into fast oxidative glycolytic fibers. Calculation on the basis of fiber length, rather than dry weight, gave an estimate of absolute enzyme changes: hexokinase was still calculated to have increased in both soleus and TA fibers, but only by 50 and 25%, respectively. Three of the OX enzymes were, on this basis, unchanged in TA fibers, but 3‐ketoacid CoA‐transferase and thiolase had still nearly doubled, whereas TA GLY enzymes had fallen about 40%. In soleus fibers, absolute levels of OX enzymes had decreased an average of 25% and GLY enzymes were marginally decreased.—Manchester, J. K.; Chi, M. M.‐Y.; Norris, B.; Ferrier, B.; Krasnov, I.; Nemeth, P. M.; McDougal, D. B., Jr.;andLowry, O. H. Effect of microgravity on metabolic enzymes of individual muscle fibers.FASEB J.4: 55‐63; 1990.