Akt substrate of 160kD (AS160), the most distal insulin signaling protein known to be important for insulin-stimulated glucose transport, becomes phosphorylated with skeletal muscle contraction. Akt, AMP-activated protein kinase (AMPK) and Ca²⁺/calmodulin-dependent kinase (CaMK)-II have been implicated in regulating AS160 and/or glucose transport. Our primary aim was to assess time-courses for contraction's effects on glucose transport and phosphorylation of Akt, AMPK, CaMKII, and AS160. Isolated rat epitrochlearis muscles were studied without or with contraction (5, 10, 20, 40, 60min). Phospho-Akt substrate (PAS) antibody was used to measure AS160 PAS-phosphorylation by quantifying the ~160kD band on PAS immunoblots (PAS-160); a separate band at 150kD (PAS-150) that responded similarly to contraction was also identified. Using specific antibodies for AS160 or TBC1D1 on immunoblots, the molecular weight of PAS-160 was found to correspond with AS160 and not TBC1D1, whereas PAS-150 corresponded with TBC1D1 and not AS160. Furthermore, supernatant of sample immunodepleted with anti-AS160 had greatly reduced PAS-160, whereas supernatant of sample immunodepleted with anti-TBC1D1 had greatly reduced PAS-150, providing further evidence that PAS-160 and PAS-150 correspond with PAS-AS160 and PAS-TBC1D1, respectively. Contraction induced transient increases in PAS-160, PAS-150, pGSK3 (an Akt substrate) and pCaMKII; glucose transport and pAMPK increases were maintained for 60min of contraction. These data suggest: 1) PAS-160 (AS160) and PAS-150 (TBC1D1) respond to contraction transiently despite sustained stimulation; 2) continual AMPK activation was insufficient for sustained increase in PAS-160 or PAS-150; and 3) sustained elevation of PAS-160 or PAS-150 was unnecessary to maintain contraction-stimulated glucose transport for up to 60min.