Recent studies have confirmed that hyperhomocysteinemia is associated with gastrointestinal diseases; however, the direct effect of homocysteine on gastrointestinal reactivity still remains unknown. The aim of this study was to demonstrate how homocysteine may affect nitric oxide mediated duodenal relaxation and whether cholinergic receptors and K+ channels take part in stimulating motility, as well as to explore whether oxidative stress is associated with homocysteine-mediated effects. Experiments were carried out on male rats, body mass 250-300 g. Two groups of animals were treated by i.p. application of saline and D,L-Hcy (0.6 μmol/g bm). After 2h of incubation, the duodenal segments were prepared for biochemical analysis and contractile response measurements in an organ bath with Tyrode’s solution. Effects of TEA (10 mmol/L) and L-NAME (30 μmol/L) on duodenal contractility in the presence of D,L-Hcy (0.6 μmol/g bm) were investigated. Elevated homocysteine levels seem to be of crucial importance for the deterioration of contractility through nitric oxide mediated relaxation, and, in part, by activation of K+ channels. Hcy showed direct promuscarinic effects, since 30 min pretreatment of rat duodenum significantly enhanced the contractile effect of increasing concentrations of ACh (10−9-10−2 mol/L). Catalase activity, superoxide dismutase, glutathione peroxidase and the total antioxidant system were reduced while the thiobarbituric acid-reactive substances level was elevated. Our data showed a consistent profile of gastrointestinal injury elicited by sulfur-containing amino acid-homocysteine. This could contribute to explain, at least in part, the mechanisms involved in human gastrointestinal diseases associated to hyperhomocysteinemia.