Horseradish is a traditional spice consumed in Eastern Europe. It is a major source of isothiocyanates; however, it contains numerous other compounds. The present study was undertaken to determine the effect of horseradish on tissue cholesterol levels in male mice (C57BL/6J) fed a cholesterol-enriched diet. Mice were randomly divided into 4 groups and received one of the following diets: control diet (C); control diet with horseradish (1%) (C + HR); cholesterol-enriched diet (0.25%) (C + Ch); and cholesterol-enriched diet with horseradish (1%) (C + Ch + HR). After 3 weeks of dietary treatment, all mice were anesthetized and total cholesterol and triacylglycerols were determined in plasma and liver. Fecal bile acids and neutral steroids were assessed as well as cecal short-chain fatty acids. The mice receiving cholesterol with horseradish (C + Ch + HR) had lower concentrations of plasma total cholesterol than those that were given a diet with cholesterol (C + Ch). Fecal bile acids were the lowest in the C group (0.41 mu mol/d). The addition of cholesterol to the diet (C + Ch) raised fecal bile acids concentration (3.65 mu mol/d), whereas horseradish supplementation (C + HR) resulted only in a slight rise of bile acid (0.77 mu mol/d) compared with the controls (0.41). In mice fed the diet containing cholesterol and horseradish (C + Ch + HR), fecal bile acid excretion was lower than in those fed the cholesterol diet (C + Ch). Horseradish increased the excretion of cholesterol and coprostanol when compared with mice fed the C diet and with cholesterol. In conclusion, horseradish lowered plasma cholesterol in mice fed the cholesterol-enriched diet. It is conceivable that the cholesterol-lowering action of horseradish could be due to the interference with exogenous cholesterol absorption.