Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 microM/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. "Flooding" tracee experiments did not overcome this problem.