Growing cultures of mutant Escherichia coli with temperature-senstive peptidyl-tRNA hydrolase were shifted to nonpermissive 4o degrees. There followed a roughly linear increase in a fraction of isolated tRNA (over 50% after 20 min) whose amino acid-accepting activity was masked until treatment with active peptidyl-tRNA hydrolase. The ionophoretic mobility of amino acid label associated with this fraction could be altered by treatment with the hydrolase, trypsin, RNAse, and alkali. The rate of accumulation of this fraction could be altered by treating the growing cells with chloramphenicol, which reduced the rate, or erythromycin, which enhanced it. It is concluded that peptidyl-tRNA dissociates from ribosomes of the mutant cells during protein biosynthesis. The primary metabolic role of peptidyl-tRNA hydrolase is to prevent the accumulation of dissociated peptidyl-tRNA, which inhibits protein synthesis. The rate of dissociation of peptidyl-tRNA from ribosomes was estimated at between 1 per 90 and 1 per 2600 peptide elongation steps in the absence of antibiotics, depending on the level of inhibition of protein synthesis. After 20 min at 40 degrees, the size distribution of peptides found on tRNA was heterogeneous, with over 74% having a molecular weight greater than 8 X 10(2). The effect of erythromycin suggests that its mechanism of action is to destabilize the peptidyl-tRNA/ribosome interaction and thereby stimulate the dissociation of peptidyl-tRNA. The mechanism of inhibition of protein synthesis by accumulating peptidyl-tRNA and reasons why peptidyl-tRNA dissociates from ribosomes are discussed in terms of the current data.