Murine L6 and human derived rhabdomyosarcoma cells were cultured at standard conditions in the presence of low (0.28 mM) and normal (9 mM) amino acid concentrations in order to re-evaluate by independent methods to what extent amino acids activate initiation of translation of protein synthesis. Independent methods were used for analysis of protein synthesis such as incorporation of radioactive amino acids into global and fractionated proteins, assessed in a two-pool model; distribution analysis of cellular RNA in density gradients of sucrose (ribosome profiles) and Western blot analyses of initiation factors with relevance for initiation of translation of cellular proteins in skeletal muscle cells (L6, RD) as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding experiments. Incorporation rate of amino acids gave incorrect results in a variety of experimental conditions, highly dependent on the concentration of tracee in the incubation medium, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated the incorporation of tyrosine into protein but not of phenylalanine. Similar problems were observed when [³⁵S] methionine was used for labeling of fractionated cellular proteins. However, methods that are entirely independent of labeled amino acids (ribosome profiles, initiation factor analyses) indicated that essential amino acids activated initiation of translation of proteins, whereas non-essential amino acids had no such effect in cultured cells. Branched-chain amino acids and glutamine, in combination with some other amino acids, stimulated also initiation of translation. Starvation/refeeding experiments in vitro agreed qualitatively with results in vivo when initiation factor states were used for comparison of synthesis in muscle cells and skeletal muscle tissue. Insulin at physiologic concentrations (100 µM/ml) did not stimulate global protein synthesis at low or normal amino acid concentrations, but did so at supraphysiologic levels (3 mU/ml) as confirmed by independent methods. Our results re-emphasize that labeled amino acids should be used with great caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in equilibrium with extra- and intracellular amino acids, even so during long term cell cultures without any restriction in tracer diffusion. "Flooding" tracee experiments did not overcome this problem.