The incorporation of [ 3 H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. The K m for puromycin is 4 μM with bacterial polyribosomes; under the same conditions, the K i for ψ-hydroxy-puromycin (6-dimethylamino-9-[3-( l -β-phenyllactylamino)-3-deoxy-β- d -ribofuranosyl] purine) is 240 μM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl- l -alanylamino)]-cyclopentyl}purine) is 1 μM. Both were found to be competitive inhibitors of puromycin. The K m for C-A-C-C-A(Phe) is 250 μM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 μM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the α-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5′-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively high K m for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.