In vitro, insulin transport across endothelial cells has been reported to be saturable, suggesting that the transport process is receptor mediated. In the present study, the transport of insulin across capillary endothelial cells was investigated in vivo. Euglycemic glucose clamps were performed in anesthetized dogs (n = 16) in which insulin was infused to achieve concentrations in the physiological range (1.0 mU/kg per min + 5 mU/kg priming bolus; n = 8) or pharmacologic range (18 mU/kg per min + 325 mU/kg priming bolus; n = 8). Insulin concentrations were measured in plasma and hindlimb lymph derived from interstitial fluid (ISF) surrounding muscle. Basal plasma insulin concentrations were twice the basal ISF insulin concentrations and were not different between the physiologic and pharmacologic infusion groups (plasma/ISF ratio 2.05 +/- 0.22 vs 2.05 +/- 0.23; p = 0.0003). The plasma/ISF gradient was, however, significantly reduced at steady-state pharmacologic insulin concentrations (1.37 +/- 0.25 vs 1.98 +/- 0.21; P = 0.0003). The reduced gradient is opposite to that expected if transendothelial insulin transport were saturable. Insulin transport into muscle ISF tended to increase with pharmacologic compared with physiologic changes in insulin concentration (41% increase; 1.37 +/- 0.18 10(-2) to 1.93 +/- 0.24 10(-2) min-1; P = 0.088), while at the same time insulin clearance out of the muscle ISF compartment was unaltered (2.53 +/- 0.26 10(-2) vs 2.34 +/- 0.28 10(-2) min-1; P = 0.62). Thus, the reduced plasma/ISF gradient at pharmacologic insulin was due to enhanced transendothelial insulin transport rather than changes in ISF insulin clearance. We conclude that insulin transport is not saturable in vivo and thus not receptor mediated. The increase in transport efficiency with saturating insulin is likely due to an increase in diffusionary capacity resulting from capillary dilation or recruitment.