The enzymatic hydrolysis of peptidyl-tRNA is described. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked α-amino groups are compared to the hydrolysis rates of N-acylaminoacyl-tRNA. It is shown that the hydrolysis rate of peptidyl-tRNA containing two peptide bonds is considerably higher than that of N-acylaminoacyl-tRNA. Moreover, the hydrolysis rate of different peptidyl-tRNA's depends on the peptide chain length. Thus, Gly 2-Phe-tRNA is hydrolyzed faster than GlyPhe-tRNA, and Gly 4Phe-tRNA is hydrolyzed faster than Gly 2Phe-tRNA. The apparent K m and v max values for Ac-Leu-tRNA are compared to those of Ala 2Leu-tRNA.