A depletion in long-chain polyunsaturated ω3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of d-[U-14C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and ω3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the ω3-depleted rats were injected 120 min before sacrifice with either a ω3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (FO) or a control medium-chain triglyceride:olive oil emulsion (OO). The content of the soleus muscle in long-chain polyunsaturated ω3 fatty acids was severely decreased in the ω3-depleted rats, and modestly albeit significantly increased after injection of FO to these animals. In stretched soleus muscles from OO-injected ω3-depleted rats, the absolute values for glycogen synthesis measured in the absence or presence of insulin were about twice higher than in normal animals. In the OO-injected ω3-depleted rats, insulin augmented the output of 14C-labelled amino acids, whilst such was not the case in normal animals. These and other findings suggest a lower catabolism of d-glucose relative to the anabolic process of glycogen synthesis and a lower availability of endogenous amino acids in the muscles of ω3-depleted rats, as compared to those of control animals. The prior injection of FO to the ω3-depleted rats restored a normal value for the paired ratio between the output of 14C-labelled amino acids and acidic metabolites, but further increased glycogen net synthesis. It is proposed, therefore, that the perturbation of d-glucose metabolism in muscles from ω3-depleted rats involves a multifactorial determinism, only some of the concerned factors being susceptible to rapid correction after enrichment of cell phospholipids in long-chain polyunsaturated ω3 fatty acids. © 2006 Elsevier Masson SAS. All rights reserved.