Abstract The transfer ribonucleic acid content of ribosomal preparations is measured by a method which employs electrophoresis in polyacrylamide gels. Peptidyl tRNA is measured separately after controlled proteolytic digestion of the ribosomes. Unlike previous methods of analysis, this method does not require radioactively labeled tRNA, is generally applicable to all types of cells, and does not require use of a cell-free system. Drug- or virus-induced modifications of tRNA binding to ribosomes can now be routinely analyzed in cellular systems under physiological conditions. The method was used with reticulocytes and with embryo skeletal muscle in the absence of inhibitors of protein synthesis and in the presence of NaF and of puromycin. In agreement with previous results, native polysomal ribosomes carry approximately two tRNA molecules, one being covalently bonded to a nascent peptidyl chain. On the other hand, single ribosomes contain only one relatively weakly bound tRNA unliked to a peptidyl chain. Brief treatment of reticulocytes with puromycin caused release from polysomes of their nascent peptide chains, apparently without causing release of the previously associated tRNA molecules from the donor ribosome sites. However, it is found here that puromycin does cause a significant release of approximately 0.4 tRNA molecules from the polysomal ribosomes, and it is suggested that this loss occurs from the ribosome acceptor sites by a noncompetitive mechanism.