Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L-[1-14C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex G-10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins.