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We have designed a method for the large scale isolation of pure and highly active subunits from yeast cytoplasmic ribosomes. The method consists of the incubation of yeast, protoplasts in the presence of 1 mM NaN3. This causes the disappearance of polysomes and the formation of 80-S ribosomes that can be dissociated in 0.5 M KCl. In this way over 90% of the ribosomes can be dissociated into subunits. The subunits are isolated by centrifugation in a B-XV zonal rotor using an equivolumetric sucrose gradient. When this separation is performed at 5 °C, dimerization of 40-S particles to 65-S particles occurs and no pure subunits can be obtained, At 10 °C, there is no dimerization and the 60-S + 40-S subunits obtained are less than 1% cross-contaminated. In poly(U)-directed protein synthesis one subunit pair polymerizes phenylalanine residues at a rate of 1.7 residue per min. About 70% of the subunits were estimated to participate in this synthesis. Subunits prepared in the absence of dithiothreitol still bound phenylalanyltRNA, but, were inactive in phenylalanine polymerization. The 60-S subunits prepared without dithiothreitol were as active as those prepared in the presence of dithiothreitol when their peptidyltransferase activity was tested. [ABSTRACT FROM AUTHOR]