BACKGROUND & AIMS: Nitric oxide (NO) is well accepted as an inhibitory neurotransmitter in the gastrointestinal tract; however, its role in the triggering of transient lower esophageal sphincter relaxations (TLESRs) in humans remains to be determined. Therefore, the effect of NG-monomethyl-L-arginine (L-NMMA), a specific NO synthase blocker, on gastric distention-induced TLESRs was investigated. METHODS: Esophageal manometry was performed using a perfused sleeve assembly. The effect of L-NMMA was evaluated on water swallow-evoked primary peristalsis (n = 8; single-blind, placebo-controlled) and on the rate of TLESRs during gastric distention (n = 8; double-blind, placebo-controlled). RESULTS: L-NMMA increased the amplitude of peristaltic pressure waves in the distal esophagus and increased peristaltic velocity in the proximal esophagus. In contrast, L-NMMA had no effect on basal lower esophageal sphincter pressure, nadir pressure, duration, and area under the curve of lower esophageal sphincter relaxation. L-NMMA significantly inhibited the increase in TLESRs during gastric distention. L-NMMA also increased the intraballoon pressure during distention. Conclusions: NO is one of the neurotransmitters involved in the reflex arc mediating the triggering of TLESRs. NO is involved in the timing of human esophageal peristalsis and may exert a tonic inhibition on the proximal stomach.