Previous measurements in lymph and adipose tissue have indicated that interstitial insulin concentrations are ∼40% lower than in plasma. Measurements of insulin in human muscle interstitial fluid have not been performed yet. We developed a new external reference technique for calibration of microdialysis catheters in situ. This technique allows correct assessments of interstitial peptide concentrations and was employed to estimate the insulin concentration in medial quadriceps femoris muscle in 11 individuals (age: 37 ± 3 yr; body mass index: 25.2 ± 1.2 kg/m²) during a two-step euglycemic hyperinsulinemic clamp. At steady-state insulin and glucose infusion, plasma glucose was 5.9 ± 0.2 mmol/l, plasma insulin was 155 ± 17 mU/l, and interstitial muscle insulin was 67 ± 19 mU/l (n = 9;P < 0.01). At a higher insulin infusion rate, the steady-state plasma insulin concentration was 379 ± 58 mU/l, and interstitial insulin concentration was 180 ± 40 mU/l (P < 0.01). The data show for the first time that high physiological and supraphysiological plasma insulin levels give 30–50% lower interstitial concentrations of insulin in the muscle. The importance of capillary delivery as a rate-limiting step for the insulin effect is suggested.