The free radical nitric oxide, generated by different types of epidermal and dermal cells, has been identified as an important mediator in various physiological and pathophysiological processes of the skin, such as regulation of blood flow, melanogenesis, wound healing, and hyperproliferative skin diseases. However, little is known about the role of NO in the human hair follicle and in hair cycling processes. Here we demonstrate for the first time that dermal papilla cells derived from human hair follicles spontaneously produce NO by measuring nitrate and nitrite levels in culture supernatants. This biomolecule is apparently formed by the endothelial isoform of nitric oxide synthase, which was detected at the mRNA and protein levels. Remarkably, basal NO level was enhanced threefold by stimulating dermal papilla cells with 5alpha-dihydrotestosterone (DHT) but not with testosterone. Addition of N-[3-(aminomethyl)benzyl]acetamidine (1400W), a highly selective inhibitor of inducible nitric oxide synthase, restrained the elevation in NO level induced by DHT. Analyses of DHT-stimulated cells at the mRNA and protein levels confirmed the expression of inducible nitric oxide synthase. These findings suggest NO as a signaling molecule in human dermal papilla cells and implicate basal and androgen-mediated NO production to be involved in the regulation of hair follicle activity.