In this study we aimed to validate the microdialysis technique for metabolic measurements in the dermal interstitial fluid. The abdominal and forearm skin was used for microdialysis in 15 healthy normal weight volunteers. The depth of the microdialysis catheter was assessed by ultrasound measurement. Structural impairment and blood flow were judged from biopsies and from laser Doppler measurements taken adjacent to the catheters. Dermal interstitial lactate and pyruvate concentrations were measured, under steady stale fasting conditions, after equilibrium calibration of each catheter in situ. The dermal Interstitial glucose concentration was estimated by means of the retrodialysis calibration method, which has previously not been evaluated for skin microdialysis. The mean catheter depth (± standard deviation) was 0.8 ± 0.3 mm. Small areas of localized bleeding, but no inflammatory reaction, was found surrounding the catheters. The perfusion in the microdialysis region was slightly increased (15–25%1. The lactate/pyruvate ratio (12 ±0.7) showed non–ischaemic values. The dermal interstitial lactate concentration was significantly higher (1171 ± 228μmol/l) than the plasma lactate (781 ± 180μmol/l), indicating an ongoing nonoxidative glucose metabolism. Retrodialysis calibration correctly estimated the dermal glucose level to be similar to that in plasma, which may indicate the usefulness of this calibration method for microdialysis studies of endogenous substrates in the dermal interstitial fluid.