Ribosomes from 8-day-regenerating rat skeletal muscle have been shown to be more active in poly(U)-directed polyphenylalanine synthesis than ribosomes from control muscle. This difference persists after salt washing of the ribosomes and does not appear to be due to the presence of ribonuclease associated with the control ribosome population. Ribosomes from control muscle were also less active than those from regenerates in the nonenzymatic binding of phenylalanyl-tRNA to ribosomes and in the peptidyltransferase reaction. Three glutamyl-tRNA isoacceptors have been isolated from 8-day-regenerating rat skeletal muscle by preparative RPC-5 chromatography of total tRNA charged with [3H]glutamic acid. The two major isoacceptors observed, tRNAgluI and tRNAgluIII, respond to the glutamic acid codons GAG and GAA, respectively. A third, minor glutamyl isoacceptor, tRNAgluII, also responds to the codon GAA. When the three isoacceptors were tested for function in a polysomal cell-free protein synthesizing system, it was found that their relative levels of utilization were essentially identical to their relative abundances. Thus, the tRNA which increases in relative amount after the induction of regeneration, tRNAgluII, is not preferentially utilized for overall muscle protein synthesis.