Background: The purpose of this study was to introduce a microdialysis technique, which makes it possible to measure the release of small inflammatory mediators into the extracellular water space in intact human skin in vivo. Using this technique, we have studied the histamine releasing properties of substance P, a putative skin mast cell releasing agent. Methods: Small hollow fibers were inserted into the upper dermis of nine healthy subjects. Each fiber was perfused with Kreb's Ringer bicarbonate buffer at a rate of 3.0 μl/min. After establishment of a baseline, each fiber was challenged intracutaneously with substance P (0 to 4 μmol/L). Samples were collected at 2-minute intervals for 18 minutes. Histamine was measured by a fluorometric method, which correlated with an enzyme immunoassay (r = 0.96). Results: Baseline dialysate histamine concentration was 1.7 ± 0.3 ng/ml. Peak histamine release after injection of vehicle, 0.5, 1, 2, and 4 μmol/L substance P was 0.0, 1.0, 6.0, 44.5, and 88.5 ng/ml, respectively (p = 0.00002). Statistically significant histamine release was demonstrated with 1.0 μmol/L substance P and greater. Most peak values were seen 2 to 4 minutes after injection. The histamine elimination showed a monoexponential decline; dialysate histamine half-life was 3.81 ± 0.28 minutes. Conclusions: This study showed that substance P releases histamine in a dose-dependent manner from intact human skin in normal subjects. We suggest that microdialysis may be a promising technique for the evaluation of mediator levels in intact human skin after intradermal injection of an inflammatory or allergenic stimulus.