The aim of this project was to study the regulation of interstitial glycerol levels in muscle in normal subjects, and to estimate interstitial muscle glycerol in obese subjects and patients with type 2 diabetes. In healthy lean subjects, microdialysis of forearm sc and muscle tissue were combined with arterial and deep venous catheterization, as well as blood flow registrations during oral glucose ingestion. In two other separate studies, obese (n = 9) vs. lean (n = 10) subjects and type 2 diabetes patients (n = 8) vs. weight-matched control subjects (n = 8) were investigated by means of muscle microdialysis during a euglycemic hyperinsulinemic clamp. Oral glucose ingestion suppressed the interstitial sc glycerol concentration by approximately 40% (P < 0.05), whereas no significant reduction of muscle interstitial glycerol was found. In contrast to the significant muscle interstitial-arterial (I-A) glycerol difference, the venous-arterial difference was small and varying throughout the oral glucose tolerance test. At steady-state hyperinsulinemia, obese subjects' interstitial muscle glycerol and I-A glycerol difference were both significantly higher than lean controls, whereas type 2 diabetes patient had interstitial muscle glycerol concentrations and I-A glycerol differences similar to those found in weight-matched controls. A significant and marked I-A glycerol difference exists in the absence of a significant venous-arterial difference, indicating that muscle glycerol cannot be taken as a marker of intramyocellular lipolysis because local turnover of muscle glycerol might be significant. The present data also suggest that, in contrast to sc tissue, muscle tissue lacks a clear antilipolytic effect of insulin. Moreover, the muscle interstitial glycerol concentration is elevated in obese patients but does not precipitate insulin resistance and type 2 diabetes.