Nutrients enhance signaling pathways involved in skeletal muscle growth through an increased rate of protein synthesis. These studies have led to an understanding of the potential role of the mammalian target of rapamycin (mTOR) in this process. However, activation of mTOR cannot account for all the stimulatory effects of nutrients. The purpose of these experiments was to examine the effect of nutrients on the cellular distribution and activation state of novel PKC isoforms (PKC- and PKC-) in the gastrocnemius of rats using modification state dependent phospho-peptide specific antibodies. The phosphorylation of PKC- on the catalytic domain autophosphorylation site (Ser⁷²⁹) was elevated during feeding and then returned to basal levels when the feeding period ended. Meal feeding augmented the phosphorylation of the downstream effectors of mTOR, namely S6K1 and 4E-BP1. In contrast, the phosphorylation of PKC- upon either the catalytic domain autophosphorylation site (Ser⁶⁴³) or activation loop site (Thr⁵⁰⁵) was unaffected. Similar results were obtained when animals were given leucine either acutely via gavage or chronically by dietary supplementation. The effect of leucine was not mimicked by injecting animals with insulin but could be induced by gavage with norleucine, a structural analog of leucine that does not increase plasma insulin concentration. Thus, rises in insulin secondary to meal feeding or leucine gavage are probably not responsible for increased phosphorylation of PKC- in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKC- in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKC- from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKC-, but not PKC- in gastrocnemius both in vivo and in vitro. Furthermore, activation of mTOR signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKC-, suggesting that multiple pathways become activated with leucine.