Pectin (PE), guar gum (GG), and psyllium (PSY) lower plasma low density lipoprotein (LDL) cholesterol concentrations in guinea pigs with different orders of magnitude by inducing defined alterations in hepatic cholesterol homeostasis (Fernandez et al. 1994. Am. J. Clin. Nutr. 59: 869-879; 61: 127-134 and 1995. J. Lipid Res. 36: 1128-1138). To further explore specific mechanisms responsible for the differences in plasma and hepatic cholesterol lowering, the effects of these fibers were evaluated on cholesterol absorption, hepatic cholesterol 7 alpha-hydroxylase activity, the rate-limiting enzyme of bile acid synthesis, and in vivo LDL transport to target specific primary and secondary mechanisms accounting for the observed responses. Fibers were fed with physiological (0.04%), low cholesterol (LC), or pharmacological high cholesterol (HC) (0.25%) levels to assess whether cholesterol intake influences plasma LDL lowering mechanisms. Intake of PE, GG, or PSY with LC or HC diets lowered plasma and hepatic cholesterol concentrations (P < 0.001). PE and PSY up-regulated 7 alpha-hydroxylase activity 3-fold with LC and PE by 5-fold with HC diets. In contrast, GG intake had no effect on 7 alpha-hydroxylase activity. Cholesterol absorption was reduced 30% by PE intake while no differences were found between control and PSY groups. GG reduced cholesterol absorption only with HC diets. Intake of PE, GG, or PSY with HC diets resulted in faster plasma LDL fractional catabolic rates (FCR) (P < 0.01) with no effect on LDL apoB flux rates (FR) or pool size, suggesting that fiber reduced LDL cholesterol concentration without decreasing the number of LDL particles. In addition to reducing LDL apoB FR, PE and PSY increased LDL FCR with HC diets while GG effects were limited to lowering LDL apoB FR. These results indicate that the distinctive reductions in hepatic cholesterol induced by PE, GG, and PSY associated with plasma cholesterol lowering result from different mechanisms specific to each fiber and that the levels of dietary cholesterol contribute to the different metabolic responses.