To investigate the mechanism by which -Hydroxy--methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4h and this was effectively attenuated by HMB (25-50µM). HMB (50µM) alone stimulated protein synthesis and this was attenuated by rapamycin (27nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70kDa ribosomal S6 kinase (p70^S6k) and initiation factor 4E binding protein (4E-BP1), and an increased association of eIF4E with eIF4G. PIF alone induced a transient (1-2h) stimulation of phosphorylation of mTOR and p70^S6k. However, in the presence of HMB, phosphorylation of mTOR, p70^S6k and 4E-BP1 was increased, the inactive 4E-BP1-eIF4E complex was reduced, while the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2 (eEF2), but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the dsRNA-dependent protein kinase (PKR) leading to phosphorylation of eukaryotic initiation factor 2 (eIF2) on the -subunit, which would inhibit protein synthesis. However, in the presence of HMB phosphorylation of PKR and eIF2alpha was attenuated and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25gkg^-1). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.