The aim of the present study was to investigate the influence of dietary-fibre (DF)-rich barley-based diets on bile acids (BA) and neutral sterols (NS) in the intestinal tract of rats. For this purpose, young male Wistar rats (n 50; ten per group) weighing about 67g were fed either a barley-free diet (control group) or diets containing 500g barley meal extrudates/kg or a barley meal–Novelose mixture (groups A–D) for 6 weeks. These barley products contained 7–24g resistant starch/100g and 7–12g (1→3),(1→4)-β-glucan/100g. More steroids were transported towards the lower parts of the intestinal tract when higher concentrations of macromolecular DF were present in the diets (P<0·001). Tauroconjugated and primary BA dominated in the contents of the small intestine. Intense enzymic conversion of BA occurred in the caecum and colon. The fermentation of DF affected indirectly the amount of formed secondary BA. The main BA present in the caecal contents were muricholic acids, hyodeoxycholic acid and cholic acid. The BA spectrum in the colonic contents was different from that in the caecum. A higher concentration of NS appeared in the intestinal contents of the groups fed the barley-based diets than in the controls (P<0·005). The microbial conversion of cholesterol to coprostanol, cholestanone and coprostanone was influenced by the amount and composition of the DF in the gut. DF in the diet may affect the concentration and spectrum of steroids in the intestinal tract. The results are relevant for the discussion of mechanisms behind the cholesterol-lowering effects of DF.