The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10 microns-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40 +/- 24 microns at 120 days of age and 44 +/- 22 microns at 350 days of age in conventional rats, and 25 +/- 17 microns (120 days) and 22 +/- 10 microns (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free; 185 +/- 73 microns, conventional: 350 +/- 115 microns). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.