We have shown previously that thoracic duct lymph insulin dynamics are well correlated with tracer-determined whole-body glucose uptake and have suggested that thoracic duct lymph insulin is representative of insulin concentration in muscle interstitial fluid. However, thoracic duct lymph is comprised of interstitial fluid from all sub-thoracic tissue beds. To investigate the relative contribution of muscle interstitial fluid to total thoracic duct lymph flow, the distribution and elimination of [14C]inulin was investigated in eight experiments with conscious dogs. Both plasma and thoracic duct lymph were measured, and a three-compartment model that was hypothesized to consist of plasma, splanchnic interstitial fluid, and muscle interstitial fluid was identified. Identifications were performed with either a bolus protocol (n = 4) or an infusion protocol (n = 4), and the predicted [14C]inulin dynamics in the splanchnic and muscle interstitial fluid compartments were compared with measured values in thoracic duct lymph. Neither compartment predicted the thoracic duct [14C]inulin dynamics; however, a model based on a percentage contribution from each tissue bed fit the thoracic data well. The relative contribution of splanchnic interstitial fluid to the total thoracic duct lymph flow averaged 78 +/- 5% for the bolus protocol and 54 +/- 5% for the infusion protocol. Thus, we conclude that in the conscious animal, approximately 25-50% of thoracic duct lymph originates from muscle interstitial fluid.