To examine the role of transcapillary insulin transport to peripheral insulin sensitivity in vivo, we performed dose-response experiments in which both plasma and thoracic duct lymph insulin and glucose utilization (Rd) were measured in conscious dogs. Euglycemic clamps (n = 22) consisted of a 3-h activation period in which insulin was infused (rates: "physiological" 3.6, 5.4, 7.2 pmol.min-1.kg-1; "pharmacological" 108 pmol.min-1.kg-1), followed by a 3-h deactivation period. [14C]inulin was also infused as a diffusionary marker. Insulin sensitivity was estimated as the ED50. When based on plasma insulin, ED50 was 480 pM. However, when calculated from lymph (i.e., interstitial) insulin measurements, ED50 was 240 pM. Thus, interstitial insulin measurements reveal that insulin sensitivity of peripheral tissues is approximately twice that estimated from plasma insulin and is similar to sensitivity reported for suppression of hepatic glucose production. Furthermore, although [14C]inulin achieved equilibrium between plasma and lymph within 180 min, within the physiological range, steady state plasma insulin was higher than insulin in lymph (306 +/- 18, 474 +/- 42, and 780 +/- 60 pM vs. 180 +/- 18, 318 +/- 12, and 504 +/- 36 pM; P less than 0.0001); plasma insulin achieved steady state faster than lymph insulin (6 +/- 1, 6 +/- 2, and 11 +/- 3 min vs. 29 +/- 4, 16 +/- 6, and 44 +/- 8 min; P less than 0.01) and disappeared faster (5 +/- 2, 7 +/- 2, and 15 +/- 6 min vs. 37 +/- 8, 32 +/- 4, and 43 +/- 9 min; P less than 0.01). The time course of lymph insulin at each dose was similar to that of Rd, and at each dose, unlike plasma insulin, lymph insulin was strongly correlated with Rd (r = 0.93 or better). At pharmacological hyperinsulinemia (plasma 35232 +/- 5250 pM, lymph 27366 +/- 4380 pM), Rd rose faster than lymph insulin and disappeared more slowly than insulin. Thus, lymph insulin data indicate that the periphery is more sensitive to insulin than previously realized from estimates based solely on plasma hormone. Furthermore, lymph insulin is proportional to Rd within the physiological but not pharmacological range of insulin, indicating that transcapillary insulin transport is rate limiting for insulin action in this range. Finally, based on in vivo lymph (i.e., interstitial) insulin measurements, peripheral tissue is almost twice as sensitive to insulin than previously realized.