Purity, integrity and protein synthesis were studied in chloroplasts isolated from barley (Hordeum vulgare L. cv. Hassan) leaf segments incubated for 20 h in the dark, a treatment which enhances subsequent senescence. Apparently, isolated chloroplasts retained enough Mg^^ for the incorporation of radioactive ainino acids into proteins. The effects of chloramphenicol, cycloheximide and nuclease and the requirement of radiant energy indicated that amino acid incorporation was due to protein synthesis on 70S chloroplast ribosomes. The electrophoretic profile of the polypeptides synthesized was similar in intact and in broken chloroplasts. Pulse-chase assays indicated that most, if not all, polypeptides labelled in assays with isolated chloroplasts were complete polypeptides, which were also synthesized in vivo. Hence the polypeptides synthesized in vivo by chloroplasts early in senescence may be accurately and reliably studied in this systém of protein synthesis by isolated chloroplasts.