The absolute concentrations of glycerol and lactate were studied with microdialysis of adipose tissue and skeletal muscle in normal-weight subjects. The basal interstitial glycerol concentration was 232 +/- 33, 96 +/- 8, and 59 +/- 6 mumol/l in fat, muscle, and arterialized plasma, respectively (P = 0.0002). This relationship was maintained during both euglycemic hyperinsulinemia, when glycerol decreased in all three compartments, and hypoglycemia, when glycerol first decreased and then increased in fat, muscle, and blood (P = 0.0001 for both). Basal interstitial lactate concentrations were similar in adipose tissue (1.1 +/- 0.2 mmol/l) and skeletal muscle (1.9 +/- 0.4 mmol/l) and higher than in arterialized blood (0.6 +/- 0.1 mmol/l, P = 0.002). During hyperinsulinemia and hypoglycemia, lactate increased (P = 0.0001) and the tissue-blood relationship was maintained (P = 0.04). In conclusion, adipose tissue and skeletal muscle mobilize glycerol and lactate at rest. Glycerol and lactate production are influenced by hyperinsulinemia and hypoglycemia in both tissues. Adipose tissue appears to be the major site of glycerol production, whereas skeletal muscle and fat may be equally important for lactate production.