Isolated pea chloroplast thylakoids ordinarily have ribosomes attached which survive sequential washes. Extensive in vivo loss of these thylakoidbound ribosomes occurred if the pea plants were placed in the dark without 02 for 2 or more hours. This loss was indicated from measurements of both the total thylakoid-bound RNA levels, and the capacity for amino acid incorporation into proteins on the addition of soluble enzymes for protein synthesis. Stroma ribosome profiles lost any indication of polysome structure due to the same anoxic treatment in vivo. The return of ribosomes to the thylakoids when plants were placed in the light in air occurred over an 8-hour time course. This return was prevented by lincomycin, spectinomycin, and chloramphenicol, indicating a requirement for protein synthesis steps in the stroma at some point in the reassociation process.The 70S chloroplast ribosomes are found both free in the stroma and attached to thyalkoid membranes in higher plants, as well as in Chlamydomonas (see 2 for references). Evidence has accumulated indicating that the membrane-bound ribosome population is engaged in the synthesis of membrane proteins (2,6,7,10,12,14,15,17). The proportion of the ribosomes bound to membranes in isolated chloroplasts was reported to be about 50%o in earlier publications (7). By taking into account the large degree of outer envelope breakage and loss of stroma in these preparations, Tao and Jagendorf (17) calculated that only 20% of the total 70S ribosomes in peas and spinach chloroplasts are present in bound form. This proportion in turn could be variable, in view of the finding by Chua et al. (6) that the number of 70S ribosomes attached to membranes in Chlamydomonas is at a maximum in the light when membrane proteins are being made, and drops precipitously when the algae are put into darkness.We are interested in studying the process by which ribosomes are attached to thylakoid membranes of higher plants. To Isolation of Chloroplasts, Membranes, and Ribosomes. Isolation of chloroplasts, stroma, and thylakoid membranes including step gradient procedures were described earlier (2). To obtain stroma ribosomes, chloroplasts were resuspended in a hypotonic buffer containing 40 mm Tris (pH 8.5), 30 mm Mg acetate, 60 mm KCI, and 5 mm ,B-mercaptoethanol, and the thylakoid membranes were removed by centrifuging at 12,000g for 10 min. The supernatant was collected, and centrifuged again at 39,000g for 30 min to remove traces of broken thylakoid membranes. The final supernatant was layered over a two-step gradient consisting of 2 ml each of 1.5 and 2.0 M sucrose in 40 mm Tris, 20 mm KCI, and 10 mm Mg acetate at pH 8.5. The ribosomes were collected by centrifuging for 3.5 h at 50,000 rpm in the Beckman 50.2 Ti rotor (L5-50 ultracentrifuge). The ribosomal pellet was resuspended and stored as described previously (2) for membrane-bound ribosomes.Measurements of Chi, Bound RNA, and Protein Synthesis. Chl was estimated by the method of Arnon (3). Thylakoid-bound RNA was measured by a p...