The subunit locations of the five enzymes associated with the fatty acid oxidation complex from Escherichia coli were studied by immunotitration and chemical modification. Antibodies raised against the purified complex caused the parallel inhibitions of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, while slightly stimulating 3-ketoacyl-CoA thiolase. All five component enzymes of the complex were inactivated by treatment with iodoacetamide. The inactivation of 3-ketoacyl-CoA thiolase was rapid, whereas the four other enzymes were inactivated at much slower, but almost equal rates. All enzymes except for 3-ketoacyl-CoA thiolase were protected against this inactivation by either NADH or crotonyl-CoA. The reaction of iodo[1-14C]acetamide with the complex in the presence and absence of NADH resulted in the differential labeling of the large subunit only. These observations together with published results (Pawar, S., and Schulz, H. (1981) J. Biol. Chem. 256, 3894-3899) lead to the suggestion that enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, cis-delta 3-trans-delta 2-enoyl-CoA isomerase, and 3-hydroxyacyl-CoA epimerase are located on the 78,000-Da subunit, whereas 3-ketoacyl-CoA thiolase is associated with the 42,000-Da subunit. Additionally, this study provides further evidence for the existence of a fatty acid oxidation (fad AB) operon that codes for the multienzyme complex of fatty acid oxidation and that is located at 85 min on the E. coli chromosome.