Previously, we have shown that in young and old men essential amino acids (EAA) stimulate anabolic signalling pathways associated with the control of muscle protein synthesis when insulin is clamped at fasting plasma concentrations. However, the degree of activation of some signalling enzymes is reduced in older men in concert with a diminished protein synthetic response, possibly contributing to ageing sarcopenia. We have now investigated whether the reduced signalling observed in the presence of AA is apparent even under hyperinsulinaemic conditions. We therefore administered intravenous mixed AA (102 mg/kg/h) with plasma insulin maintained at 150 μIU/ml in 8 young and 8 older men (mean 24 and 67 y, respectively). Quadriceps muscle was sampled using local anaesthesia at baseline and after 2.5 h. Phosphorylation was assessed by Western blotting for protein kinase B (PKB) thr308, glycogen synthase kinase 3β (GSK3β) ser9, eukaryotic initiation factor 2α (eIF2α) ser51, mammalian target of rapamycin (mTOR) ser2448, p70s6K1 thr389, 4E-binding protein-1 (4E-BP1) thr37/46. Eukaryotic initiation factor 4E (eIF4E) proteins were extracted for analyses of eIF4E associated with eIF4G using m7¬GTP sepharose. Phosphorylation of PKB and GSK3β increased in young and old men (40-fold; P<0.01 vs 10-fold; P<0.05 and 3.9-fold; P<0.05 vs 2.1-fold; P<0.05, respectively) although significantly more in younger men (P<0.05). Phophorylation of eIF2α decreased significantly only in younger men, 57 % (P<0.05) compared with 24 % in older men. Phosphorylation of mTOR did not change in either group, nor did amounts of eIF4E associated with eIF4G. The phosphorylation of p70s6K1 was increased in both groups (24-fold; P<0.01 vs 7-fold; P<0.05) although to a greater extent in younger men (P<0.05). The phosphorylation of 4E-BP1 was increased to a similar extent in both young and old men (2.8-fold vs 3-fold; both P<0.05, respectively). The reduction in eIF2α phosphorylation in the younger men suggests that the exchange of GTP on eIF2B is modulated by feeding, an affect diminished with age. Coupled with the reduced GSK3β phosphorylation in older men, this might reduce cellular eIF2B-GTP concentrations and thus Meti tRNA recycling. The observed differences in phosphorylation of signalling molecules in older men (possibly related to dysregulated insulin receptor signalling) might explain age-related differences in the normal food stimulated increase in protein synthesis and conceivably even the blunting of protein breakdown observed in skeletal muscle. We provide new information concerning AA and insulin-induced signalling events in human skeletal muscle and show that hyperinsulinaemia does not rejuvenate the blunted anabolic signalling observed in older men.