--- - |2+ Key points It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic‐euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased muscle p‐eIF2αSer51 and increased muscle protein synthesis. Abstract It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We tested this hypothesis by measuring muscle p‐mTORSer2448, p‐p70S6KThr389 and p‐eIF2αSer51, as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45 g protein kg−1 fat‐free mass (containing 0.0513 g leucine kg−1 fat‐free mass) or a control drink (n = 14) or 0.0513 g leucine kg−1 fat‐free mass or a control drink (n = 14) during a hyperinsulinaemic‐euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p‐mTORSer2448 and p‐p70S6KThr389 by >50% (all P < 0.05) but had no effect on p‐eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased (by ∼35%) p‐eIF2αSer51 and increased (by ∼100%) MPS (all P < 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation. - The Journal of Physiology, Volume 596, Issue 19, Page 4681-4692, 1 October 2018.