In this study, we have investigated the hypothesis that an exercise protocol designed to repeatedly induce a large dependence on carbohydrate and large increases in glycolytic flux rate would result in rapid increases in the principal glucose and lactate transporters in working muscle, GLUT4 and MCT4, respectively, and in hexokinase (HEX) activity, the enzyme used to phoshorylate glucose. Transporter abundance and HEX activity were assessed in homogenates by Western blotting and quantitative chemilumenescence and fluorometric techniques, respectively, in samples of tissue obtained from the vastus lateralis in 12 untrained volunteers (VO_2peak=44.3±2.3 ml.kg.min ) prior to cycle exercise at repetitions 1(R1), 2(R2), 9(R9) and 16 (R16). The 16 repetitions of the exercise were performed for 6 min at ~90% VO_2peak, once per hour. Compared to R1, GLUT4 increased (P0.05) by 28% at R2 and remained elevated (P0.05) at R9 and R16. For MCT4, increases (P0.05) of 24% were first observed at R9, which persisted at R16. No changes were observed in GLUT1 and MCT1 or in HEX activity. The approximate 17—24 fold increase (P0.05) in muscle lactate observed at R1 and R2 was reduced (P<0.05) to a 11 fold increase at R9 and R16. It is concluded that an exercise protocol designed to strain muscle CHO reserves and to result in large increases in lactic acid results in a rapid up-regulation of both GLUT4 and MCT4.