Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyperplastic growth and tissue morphogenesis in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian Target of Rapamycin (mTOR), the cytosolic, Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate limiting step in leucine oxidation is catalyzed by the branched chain -keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1-subunit at Ser293 (S293), which inactivates the complex. Because the -ketoacid of leucine (KIC) regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phospho-peptide specific antibody recognizing this site was developed and characterized. Phospho-S293 immunoreactivity in liver corresponded closely to diet induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylation in adipose tissue and gastrocnemius (which is mostly inactive in vivo) was similar. This suggests that BCKD complex in epidydimal adipose tissue from food-deprived rats is mostly inactive (unable to oxidized leucine) as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pSer293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (T389, the phosphorylation site leading to activation) in adipose tissue was maximal at dose of leucine that increased plasma leucine approximately 3 fold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation, than in the activation of the leucine signal to mTOR.