Previous studies have described the magnitude and time course by which several genes are regulated within exercising skeletal muscle. These include interleukin-6 (IL-6), interleukin-8 (IL-8), heme oxygenase-1 (HO-1), and heat shock protein-72 (HSP72), which are involved in secondary signaling and preservation of intracellular environment. However, the primary signaling mechanisms coupling contraction to transcription are unknown. We hypothesized that exercise-induced nitric oxide (NO) production is an important signaling event for IL-6, IL-8, HO-1, and HSP72 expression in muscle. Twenty healthy males participated in the study. By real-time PCR, mRNA levels for 11 genes were determined in thigh muscle biopsies obtained 1) before and after 2 h knee extensor exercise without (control) and with concomitant NO synthase inhibition (nitro-L-arginine methyl ester, L-NAME, 5 mg·kg^-1); or 2) before and after 2 h femoral artery infusion of the NO donor nitroglycerin (NTG, 1.5 µg·kg^-1·min^-1). L-NAME caused marked reductions in exercise-induced expression of 4 of 11 mRNAs including IL-6, IL-8, and HO-1. IL-6 protein release from the study leg to the circulation increased in the control but not in the L-NAME trial. NTG infusion significantly augmented expression of the mRNAs attenuated by L-NAME. These findings advance the novel concept that NO production contributes to regulation of gene expression in muscle during exercise. Subsequently, we sought evidence for involvement of AMP-activated kinase or nuclear factor kappa B, but found none.--Steensberg, A., Keller, C., Hillig, T., Frøsig, C., Wojtaszewski, J. F. P., Pedersen, B. K., Pilegaard, H., Sander, M. Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle.