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A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The effect of the car mutation on chloroplast ribosome function was demonstrated with an in vitro system incorporating amino acids especially developed to assess activity of 70S chloroplast ribosomes. In an S-30 extract containing both 70S chloroplast and 80S cytoplasmic ribosomes, low concentrations of Mg(++) and spermidine favored 80S ribosome activity, and high concentrations activated 70S ribosomes and reversibly inactivated the 80S component. Under conditions favoring chloroplast ribosome activity, carbomycin inhibited incorporation by an S-30 extract, and by purified 70S ribosomes from wild-type but not from car cells. These results show that cytoplasmic genes are directly involved in chloroplast ribosome function and they suggest that the car gene product is a ribosomal protein; the results further strengthen the evidence that the cytoplasmic linkage group is located in chloroplast DNA.