We previously reported a striking similarity between the dynamics of both glucose turnover and thoracic duct lymph insulin during euglycemic clamps (J Clin Invest 84:1620, 1989), which suggested that transendothelial insulin transport (TET) is rate-limiting for insulin action in vivo. Thoracic duct lymph, however, is primarily derived from insulin-insensitive tissues, which raises questions as to the physiological significance of this relationship. The relationship between glucose turnover and TET was thus examined in insulin-sensitive tissues by the simultaneous measurement of insulin in plasma, thoracic duct lymph, and hindlimb lymph during euglycemic clamps in normal anesthetized dogs (n = 8). Clamps consisted of two 3-h phases: a 0.6 mU · min⁻¹ · kg⁻¹ insulin infusion (activation phase) followed by termination of the insulin infusion (deactivation phase). Lymph insulin was < plasma insulin during both phases (P < 0.01) with steady-state hindlimb (120 ± 12 pM) and thoracic duct lymph insulin (138 ± 12 pM) 38 and 45%, respectively, lower than steady-state plasma insulin (222 ± 24 pM) at the end of the activation phase (P < 0.05). Also, the rate of increase of lymph insulin was slower than plasma insulin during hormone infusion; half-time to steady-state was 8.8 ± 2.0 min for plasma insulin, but longer for thoracic (25.8 ± 3.5) and hindlimb lymph insulin (40.7 ± 5.7 min). A very close relationship was observed during activation between the rate of increase of glucose uptake (R_d) and the increase in hindlimb lymph insulin (r² = 0.92); this relationship was weaker for thoracic lymph (r² = 0.74) and much weaker between glucose uptake and plasma insulin (r² = 0.35). These data support the concept that interstitial insulin (represented by hindlimb lymph) is the signal that determines glucose uptake by insulin-sensitive tissues and that the rate of increase of glucose uptake is determined by transendothelial insulin transport into insulin-sensitive tissue. Also, during activation, hindlimb lymph insulin was a very strong predictor of the rate of suppression of hepatic glucose output (HGO) (r² = 0.96), and the correlation with HGO was stronger than that for thoracic lymph (r² = 0.85). The evidence that the rate of increase of R_d and the rate of suppression of HGO during insulin infusion are very strongly predicted by the time course of insulin in hindlimb lymph is consistent with the single-gateway hypothesis: the insulin transport rate across endothelium in insulin-sensitive tissue (skeletal muscle) determines the rate of glucose utilization and the suppression of hepatic glucose output. It is suggested that there is a yet-undefined signal that controls HGO generated at the level of insulin-sensitive peripheral tissues.