In the present study the microdialysis technique was evaluated in an isolated autoperfused dog fat pad. Concentrations of glucose, lactate, and glycerol were measured in interstitial fluid by microdialysis and simultaneously in arterial and adipose venous plasma. Adipose tissue blood flow was measured by both 133Xe washout and timed weighing of venous blood. Metabolite concentrations in adipose venous plasma calculated from interstitial and arterial metabolite concentrations and 133Xe washout were positively correlated with measured venous concentrations (glucose: r = 0.95, lactate: r = 0.92, glycerol: r = 0.81). Calculated and measured venous plasma concentrations did not differ for either glucose or lactate, but for glycerol, calculated concentration was on average 76% of measured concentration. Metabolite exchanges (Fick's principle) calculated from interstitial metabolite concentrations were positively correlated with measured exchanges only for lactate (r = 0.69). In conclusion, metabolite concentrations in adipose venous plasma can be calculated from microdialysis measurements with greater accuracy for glucose and lactate than for glycerol. The precision, however, is too low to allow calculation of metabolite exchange when arteriovenous metabolite differences are low.