We investigated the effects of hydrogen peroxide $(H_2O_2)$ on relaxation of the cat lower esophageal sphincter (LES). Vasoactive intestinal peptide (VIP) caused dose-dependent relaxation of LES, and $H_2O_2$ reduced VIP-induced relaxation. Relaxation was also attenuated by pertussis toxin (PTX), indicating a Gi/o component. VIP treatment increased $[^{35}S]GTP{gamma}S$ binding to Gs and Gi3 protein, but not to Go, Gq, Gi1 or Gi2. This increase in Gs or Gi3 binding was reduced by $H_2O_2$. However, the relaxation induced by sodium nitroprusside (SNP), 3-morpholino sydnomine (SIN-1), 8-br cGMP (cGMP analog), forskolin (adenylate cyclase activator), and dibutyryl-cAMP (a stable cAMP analog) was not reduced by $H_2O_2$. These data suggest that $H_2O_2$ inhibits VIP-induced relaxation via a Gi-dependent pathway, perhaps by inhibiting the activation of $G_{i3}$ or Gs downstream of the VIP receptor and independent of cAMP or NO-cGMP signaling.