Sarcopenia is an age-associated disorder that causes loss of skeletal muscle mass, particularly in type II (fast-twitch) muscle fibers. This loss in muscle mass can cause disability, reductions in the quality of life, and can contribute to the development of other more life-threatening morbidities and even death. Researchers have utilized muscle overloading and ergogenic aids, such as whey protein and essential amino acids (specifically leucine), in rats and humans in attempts to reduce or attenuate these losses as part of a primary prevention strategy. Unfortunately, there is also a loss of overload-induced growth capacity in aged fast-twitch skeletal muscle. However, no studies have explored the potential synergistic effect of leucine supplementation on overload-induced skeletal muscle growth in aged animals. To that end, the purpose of this study was to examine the effects of dietary leucine supplementation on protein translational signaling and hypertrophy in the overloaded fast-twitch skeletal muscles of aged animals. It was hypothesized that supplementing a standard chow diet with 5% leucine would enhance muscle hypertrophy in overloaded fast-twitch plantaris muscles of aged (33-month old) rats to levels observed in young adult (8-month old) rats. It was also hypothesized that 5% dietary leucine supplementation would enhance protein translational [70 kDa ribosomal protein S6 kinase (p70s6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 kinase (eEF2k), and eEF2] signaling in the overloaded fast-twitch plantaris muscles of aged rats to levels observed in young adult rats. Young adult and old male Fisher³�� x Brown Norway F1 Hybrid (FBN) rats underwent a 1-week unilateral plantaris muscle overload via tenotomy of the synergistic gastrocnemius muscle. Within each age group, animals were matched for body weight and separated into either a dietary leucine supplementation group (additional 5% leucine content in place of 5% of the carbohydrate content in normal rat chow starting 2 days prior to, and throughout, the overload intervention; n = 7/age group) or placebo group (normal rat chow; n = 6/age group). No differences in daily calorie consumption were observed between the placebo vs. leucine groups within each age group. Plantaris muscles were harvested at the end of the overload period. Dietary leucine enrichment significantly (p [equal or less than] 0.05) enhanced overload-induced fast-twitch plantaris muscle hypertrophy in old, but not in young adult, animals. Additionally, western blotting analyses revealed that phospho-p70S6k (Thr389) and phospho-rpS6 (Ser235/Ser236) were significantly lower in old vs. young overloaded muscles under placebo conditions, but leucine partially restored both phospho-p70S6k and phospho-rpS6 in old overloaded muscles to that of young adult overloaded muscles. Overload significantly increased eEF2k phosphorylation in young, but not in old animals, and leucine supplementation had no affect on eEF2k phosphorylation in either age group. Overload significantly increased total eEF2 content and decreased inhibitory eEF2 phosphorylation (Thr56; normalized to total eEF2) in young adult muscles regardless of leucine supplementation. Total eEF2 content was unaffected by overload in old placebo muscles, but leucine supplementation in old animals non-significantly (p = 0.09) restored the overload-induced increase in total eEF2 content. These novel findings indicate that a leucine-enriched diet may potentially enhance overload-induced growth of aged fast-twitch muscle, in part by enhancing pathways known to stimulate protein synthesis.M.S