Reactive oxygen species (ROS) have been implicated in the pathogenesis of many colonic diseases. Mucus is the colon's first line of defence against luminal agents. This study has therefore characterised ROS action on colonic mucus secretions. ROS were produced using peroxide-based systems of different concentrations. The effects of these systems were tested on native colonic mucus gels, isolated colonic mucins, and in vivo models. Colonic mucus gels were resistant to ROS breakdown. Mucins were susceptible to ROS attack, causing loss of terminal sugars and protein and mucin fragmentation. The in vivo thickness of the mucus barrier was reduced by up to 50% by ROS (above 5 mM peroxide). A 5 mM peroxide caused a significant increase in resting mucus thickness of ca. 15%. All ROS-generating systems caused mucosal damage once the loosely adherent mucus had been removed. As native mucus gel is more resistant to ROS damage than purified mucin, nonmucin components of mucus may have extensive ROS-scavenging properties. Low levels of luminal colonic ROS increase the protection afforded by the mucus barrier in vivo. Higher levels of ROS significantly reduce this protection. In vitro modeling of mucus degradation by ROS does not necessarily correlate with the dynamic, in vivo situation. © 2007 Elsevier B.V. All rights reserved.