The component of the flow- or agonist-dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium-dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9-derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO- and prostacyclin (PGI(2))-independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser-Doppler fluxmetry (LDF). The inhibitors of NOS, N(omega)-monomethyl-L-arginine (L-NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh-induced increase in LDF (t-test, P<0.05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L-NMMA- and diclofenac-pretreated site. In addition, sulfaphenazole did not attenuate the ACh-induced vasodilatation in either site. We conclude that a NO- and PGI(2)-independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh-induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9-derived metabolite.