In mammalian models of cirrhosis, plasma ammonia concentration increases, having numerous adverse effects, including sarcopenia. The objective of this study was to identify differences between avian and mammalian myogenic response to applied ammonia and glutamine. Primary chicken breast and thigh, primary rat, and C2C12 myotubes were treated with ammonium acetate (AA, 10 mM) or glutamine (10 mM) for 24 h and compared with sodium acetate (10 mM) and untreated controls. Myostatin mRNA was significantly higher in C2C12 and rat myotubes treated with AA compared with glutamine and controls (P < 0.01), whereas myostatin was unchanged in chicken myotubes. AA-treated C2C12 myotubes had significantly higher glutamine synthetase (GS) mRNA expression compared with controls, but GS protein expression was unchanged. In contrast, GS mRNA expression was unchanged in thigh myotubes, but GS protein expression was significantly higher in AA-treated thigh myotubes (P < 0.05). In both breast and thigh myotubes, intracellular glutamine concentration was significantly increased in AA- and glutamine-treated myotubes compared with controls but was only increased in glutamine-treated C2C12 and rat myotubes (P < 0.05). Glutamine concentration was significantly higher in all treatment media collected from avian myotube cultures compared with both C2C12 and rat media (P < 0.01). Myotube diameter was significantly larger in avian myotubes after treatment with both AA and glutamine (P < 0.05). C2C12 and rat myotubes had a significantly smaller myotube diameter after AA treatment (P < 0.001). Altogether, these data support species differences in skeletal muscle ammonia metabolism and suggest that glutamine synthesis is a mechanism of ammonia utilization in avian muscle.