Abstract Electron microscopic examination of growing spheroplasts prepared from Saccharomyces cerevisiae revealed ribosome-like particles of 150 A diameter aligned along the outer mitochondrial membrane. Ribosome-like particles were also seen associated with membranes of the endoplasmic reticulum and the outer nuclear membrane. Vacuolar and plasma membranes, however, did not appear to have attached particles. When incubated in an amino acid-incorporating system containing pH 5 fraction, mitochondria isolated in the presence of 10 mm Mg2+ were capable of incorporating [3H]leucine into trichloroacetic acid-insoluble material. About 80 % of the incorporation was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis, indicating that the majority of the amino acid-incorporating activity was due to cytoplasmic ribosomes associated with mitochondria. Mitochondria-bound cytoplasmic ribosomes were capable of vectorial release of nascent chains as shown by the distribution of puromycin-discharged polypeptides: half remain associated with the mitochondria and were releaseable by detergent, and half were released directly into the medium. These results are comparable to data reported for the distribution of puromycin-released nascent polypeptide chains with preparations of rough endoplasmic reticulum from rat liver. About one-third of the mitochondria-associated cytoplasmic ribosomes could be released by incubating mitochondria in a buffer of high ionic strength. The remaining two-thirds, however, were released only after reacting their nascent polypeptide chains with puromycin. This result suggests a role for the nascent polypeptide chains in the attachment of cytoplasmic polysomes to mitochondria. Cytoplasmic polysomes can be released intact from isolated mitochondria by extraction with Triton X-100.