The gastroduodenal mucus layer is considered the primary mucosal protective barrier, especially important in the maintenance of a mucosal pH gradient. Thus, the measurement of the mucus layer thickness in various disease states could advance our understanding of gastroduodenal pathophysiology. We present a novel method for measuring the mucus layer in endoscopic biopsy material and compare layer thickness in Helicobacter pylori (HP)-negative and HP-positive specimens. Endoscopic biopsies were obtained from 17 patients with gastroduodenal mucosa harboring HP and from 15 patients without current HP colonization. The thickness of the mucus layer was measured in fresh specimens by the phase-contrast dark-field microscopy technique. In patients with confirmed HP infection, the thickness of the mucus layer (mean +/- SD) was 0.093 +/- 0.033 mm in duodenal, 0.085 +/- 0.027 mm in antral, and 0.105 +/- 0.033 mm in corporal mucosa. In patients without concomitant HP colonization, the thickness of the mucus gel was 0.162 +/- 0.045 mm, 0.175 +/- 0.067 mm, and 0.161 +/- 0.064 mm in duodenum, antrum, and corpus, respectively. The differences between the means were statistically significant (p less than 0.001 for the duodenal, p less than 0.001 for antral, and p less than 0.01 for corporal mucosa). This study suggests that colonization of the gastroduodenal mucosa by HP impairs the mucus layer covering the surface epithelium. This mucus layer impairment may lead to mucosal injury with subsequent development of inflammation and, possibly, peptic ulcer disease.