Background & Aims Duodenal mucus and bicarbonate secretion appear to play an essential role in the protection of the duodenum. The aim of this study was to examine duodenal bicarbonate and mucus secretion and the effects of cyclooxygenase inhibition, nitric oxide synthase inhibition, and luminal acid. Methods Duodenal mucus gel thickness was measured using microelectrodes during intravital microscopy in anesthetized rats. Bicarbonate secretion was measured using back-titration. Results A continuous layer of mucus with a mean thickness of 284 ± 11 μm (n = 35) and a mean alkaline secretion of 0.18 ± 0.01 μmol · cm⁻² · min⁻¹ were found in untreated animals. Indomethacin decreased both mucus and bicarbonate secretion by about 35%. NO synthase inhibition with N^ω-nitro-l-arginine reduced mucus secretion by about 21% but increased bicarbonate secretion by 39%. Exposure of the mucosal surface to 10 mmol/L HCl increased mucus secretion by 44% and bicarbonate secretion by 22%. Conclusions The duodenal mucus layer is continuous. It can be easily removed, and new secretion can be followed. Duodenal mucus secretion is strongly stimulated by luminal acid and endogenous prostanoids and less markedly by NO, whereas bicarbonate secretion is stimulated by acid and endogenous prostanoids and inhibited by endogenous NO