The interaction of insulin with metformin on muscle glucose metabolism was examined in the perfused rat hindquarter. Glucose, lactate, and insulin were measured at the inflow to and outflow from the hindquarter, which was perfused with human erythrocytes suspended in a Kreb's-Ringer albumin buffer for 120 min. Perfusions were performed with no additions (I) and with insulin infusions targeted to concentrations of 175 (II) and 350 pmol/l (III) as well as infusions targeted to levels of 0 (IV), 70 (V), and 175 pmol/l (VI) but in the presence of metformin (90 µg/ml). In the presence of metformin, identical infusion rates of insulin yielded higher insulin concentrations, namely 283 ± 19 vs. 202 ± 31 pmol/l for VI and II, respectively (P < 0.05). Glucose uptake (GU) increased correspondingly to 79.8 ± 0.8 in VI from 60.8 ± 2.1 for IV and 50.1 ± 1.3 for II and 46.1 ± 2.7 mg/120 min for I (P < 0.05). This enhanced GU was matched by increasing insulin levels using only a higher rate of its infusion (III): GU of 70.2 ± 2.4 mg/120 min with insulin of 334 ± 26 pmol/l (P > 0.05). The simple concurrent presence of metformin and insulin [matching insulin concentrations in II rather than infusion rates (IV)] demonstrated no additonal effect on GU above that of metformin. The synergistic effects of metformin and insulin could thus be explained by a metformin-mediated decrease in the extraction of insulin by the hindquarter (4.8 ± 0.4% vs. 8.6 ± 0.9%, P < 0.05). This increases interstitial insulin (and, in a closed system, perfusate insulin), which acts on cell surface receptors to increase glucose uptake. The results demonstrate that the extracellular insulin concentration, rather than insulin internalization and degradation, is the primary determinant of insulin action on GU in muscle and that changes in tissue insulin extraction may alter local concentrations and, therefore, systemic insulin sensitivity. This provides both a physiological mechanism and a possible therapeutic target for improving insulin sensitivity.