In an attempt to clarify the role of the nucleolus in protein synthesis in eukaryotic cells, incorporation of l-[ 14C]leucine into the nucleoli of mouse ascites tumor cells both in vivo and in vitro was studied. 1. 1. The amino acid was incorporated into proteins of the nucleoli in vivo through a cycloheximide-sensitive reaction system. The specific activity of the incorporation (counts/min per mg protein) in this subnuclear organelle increased rapidly and exceeded that of the extranucleolar nuclear fraction during a pulse-labeling period of 5–10 min. 2. 2. Labeled proteins continued to accumulate in all nucleolar components analyzed in sucrose gradients during the first 1 h. With longer labeling periods, the radioactivity began to fall in the heavier fraction without any significant change in lighter ones. Finally 24 h after administration of l-[ 14C]leucine, the labeled proteins remained solely in the top area of the sucrose gradient. 3. 3. Although the incorporation in vivo was blocked more than 90% by cycloheximide (15 mg/kg body weight), there was no diminution and shift of the protein level among these nucleolar components until 30 min after the cessation of protein synthesis caused by this antibiotic. 4. 4. The nucleoli, deprived of ribonucleoprotein particles in the heavier fractions by treatment with actinomycin D (2 mg/kg body weight) for 10 h, were still capable of incorporating the amino acid into the lighter fractions in vivo. 5. 5. There was incorporation of l-[ 14C]leucine into the isolated nucleoli in vitro, but the reaction was insensitive to an addition of cycloheximide (300 μg/ml) and ribonuclease (EC 2.7.7.16) (10 μg/ml) and to an omission of ATP, ATP-regenerating system, GTP and Mg 2+. It was also found that removal of the amino-acid mixture caused enhancement of the incorporation by factors of 4–5. A GTP-dependent incorporation of [ 14C]leucyl-tRNA was not observed in the nucleoli in vitro. These extraordinary properties were never detected in the microsome fraction of the tumor cells. 6. 6. These results strongly suggest a possibility that the nucleoli of mouse ascites tumor cells are not a site of protein synthesis via an 80-S ribosomal system; rather it appears likely that the proteins in the nucleoli, if not all, originate from other cellular components, which have not yet been identified.