Chemically altered hemoglobin-based oxygen carriers have been developed as prototype blood substitutes. Such molecules may affect numerous biological processes, since free hemoglobin scavenges nitric oxide (NO). Diaspirin cross-linked hemoglobin (DCLHb) is a chemically cross-linked molecule, which has a pressor effect on blood pressure, mainly mediated by NO scavenging. However, the effects of DCLHb on the gastrointestinal and biliary motility have not been reported. This study was conducted to investigate the effects of DCLHb on the duodenal and biliary motility and determine if the underlying mechanism involves a NO pathway. Blood pressure, duodenal, sphincter of Oddi and gallbladder motility and trans-sphincteric flow were recorded in anesthetized Australian Brush-tailed possums. The effects of intravenously administered DCLHb (10% solution) or oncotically matched human serum albumin (HSA) solution on these parameters were investigated. To determine the involvement of a NO-mediated pathway in these effects, animals were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) before DCLHb or HSA was given. DCLHb increased blood pressure and duodenal contraction frequency and slowed trans-sphincteric flow compared with the HSA control. The effects of DCLHb on blood pressure and trans-sphincteric flow were immediate and transient, whereas the effect on duodenal contraction frequency was delayed and long-lived. Pretreatment with L-NAME alone increased blood pressure and duodenal contraction frequency and slowed trans-sphincteric flow. DCLHb-induced changes were not evident in the presence of L-NAME. These findings suggest that DCLHb affects duodenal and trans-sphincteric flow predominantly by NO scavenging.