To evaluate the effect of passive muscle shortening and lengthening (PSL) on the transcapillary exchange of glucose, lactate, and insulin in the insulin-stimulated state, microdialysis was performed in rat quadriceps muscle. Electrical pulsatile stimulation (0.1 ms, 0.3-0.6 V, 1 Hz) was performed on the sciatic nerve in one leg to induce passive tension on the quadriceps during a hyperinsulinemic-euglycemic clamp (10 mU · kg¹ · min¹). In the non-insulin-stimulated (basal) state, the muscle arterial-interstitial (A-I) concentration difference of glucose was 1.6 ± 0.3 mM (P < 0.01). During insulin infusion, it remained unaltered in resting muscle (1.3 ± 0.3 mM) but diminished during PSL. In the basal state there was no A-I concentration difference of lactate, whereas in the insulin infusion state it increased significantly and was significantly greater in moving (2.8 ± 0.5 mM, P < 0.01) than in resting muscle (0.7 ± 0.4 mM). The A-I concentration difference of insulin was equal in resting and moving muscle: 86 ± 7 and 100 ± 8 µU/ml, respectively. Muscle blood flow estimated by use of radiolabeled microspheres increased during PSL from 17 ± 4 to 34 ± 6 ml · 100 g¹ · min¹ (P < 0.05). These results confirm that diffusion over the capillary wall is partly rate limiting for the exchange of insulin and glucose and lactate in resting muscle. PSL, in addition to insulin stimulation, increases blood flow and capillary permeability and, as a result, diminishes the A-I concentration gradient of glucose but not that of insulin or lactate.