We hypothesized a differential activation of the anabolic signaling proteins protein kinase B (PKB) and p70 S6 kinase (p70S6K) and subsequent differential stimulation of human muscle protein synthesis (MPS) after dynamic shortening or lengthening exercise. Eight healthy men [25 ± 5 yr, BMI 26 ± 3 kg/m-2 (means ± SD)] were studied before and after 12 min of repeated stepping up to knee height, and down again, while carrying 25% of their body weight, i.e., shortening exercise with the "up" leg and lengthening exercise with contralateral "down" leg. Quadriceps biopsies were taken before and 3, 6, and 24 h after exercise. After exercise, over 2 h before the biopsies, the subjects ingested 500 ml of water containing 45 g of essential amino acids and 135 g of sucrose. Rates of muscle protein synthesis were determined via incorporation over time of [1-13C]leucine (<or=6 h after exercise) or [1-13C]valine (21-24 h after exercise) and phosphorylation of signaling proteins by Western analysis. PKB and p70S6K phosphorylation increased ∼3-fold after 3 h and remained elevated at 6 and 24 h. After exercise, rates of myofibrillar and sarcoplasmic protein synthesis were unchanged over the period including exercise and 3 h of recovery but had increased significantly at 6 (∼3.0- and 2.4-fold, respectively) and 24 h (∼3.2- and 2.0-fold, respectively), independently of the mode of exercise. Short-term dynamic exercise in either shortening or lengthening mode increases MPS at least as much as resistance exercise and is associated with long-term activation of PKB and p70S6K.