A majority of the total protein synthesis in host cell cytoplasm is inhibited by cycloheximide. Because leghemoglobin represents a large proportion of total cellular protein in the nodule, this observation suggests that leghemoglobin may be translated on the 80Stype ribosomes. Analysis of the nascent peptides isolated from free and membrane-bound polysomes showed that free polysomes contain more immunoreactive material against antibodies to leghemoglobin as compared to that of membrane-bound polysomes. When free and membrane-bound polysomes were incubated in a wheat embryo cell-free protein-synthesizing system, a larger percentage of the released polypeptides from free polysomes was found to be immunoreactive. Similar results were obtained by translation in vitro of the poly(A)containing mRNA isolated from free and membrane-bound polysomes. For immunocytochemical localization of leghemoglobin, the antibodies were conjugated with ferritin. Antibody conjugates were strictly localized in the host cell cytoplasm and ad*acent to the outer surface of the membrane surrounding the bacteroids. Ferritin was not found on the inner surface of the membrane or within the membrane sac. These data suggest that leghemoglobin is synthesized preferentially on the free polysomes in the host cell cytoplasm, directed by a poly(A)containing 9S mRNA that is most likely of plant origin, and that its location is restricted to the host cell cytoplasm. Although the precise function of leghemoglobin (Lb) in symbiotic nitrogen fixation is not known, its presence has been directly correlated with the nitrogen fixation capacity of nodules (1-3). Since the first observation (4) that Lb is confined to cells that contain bacteroids, there has been a continuous controversy over its intracellular site of synthesis and location in the cell. In order to assign a physiological role to Lb it is important to find its intracellular location. Based on autoradiographic (5) and peroxidase staining (6) techniques for detecting heme, it has been concluded that Lb is located within the membrane sacs surrounding the bacteroids. However, later it was shown by x-ray microprobe analysis that plant cytoplasm contains more iron than the sac enclosing the bacteroids (7) and thus Lb may be present in the cytoplasm. These studies (5-7) were based on the indirect methods of localization of Lb as heme or iron. Since bacteria are devoid of any Lb (4, 6), although they appear to be the site of heme synthesis (8), the presence of iron or heme may not be a useful criterion for its intracellular localization. It has been suggested that apo-Lb is synthesized in the cytoplasm of plant cells (9).We have shown (10) that this protein is encoded by a poly(A)-containing 9S mRNA that is associated with 80S-type plant ribosomes in vivo. We present further evidence that Lb is preferentially synthesized on free polysomes in the host cell cytoplasm. Using monospecific antibodies conjugated with ferritin, we found that Lb is strictly localized in the plant cell cytoplasm and does...