Fibrates are hypolipidemic drugs that affect the expression of genes involved in lipid metabolism by activating peroxisome proliferator-activated receptors (PPARs). Fibrate treatment causes adverse changes in biliary lipid composition and decreases bile acid excretion, leading to an increased incidence of cholesterol gallstones. In this study, we investigated the effect of fibrates on bile acid synthesis. Ciprofibrate and the PPARalpha agonist Wy14,643 decreased bile acid synthesis in cultured rat hepatocytes and suppressed cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, paralleled by a similar reduction of the respective mRNAs. Treatment of rats with 0.05% (wt/wt) ciprofibrate decreased cholesterol 7alpha-hydroxylase enzyme activity and mRNA. The functional involvement of PPARalpha in the suppression of both enzymes was proven with the use of PPARalpha-null mice. In wild-type mice, ciprofibrate reduced cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase enzyme activities and mRNA. The decrease in mRNA of both enzymes is regulated transcriptionally and posttranscriptionally, respectively, resulting in a decline in the output of fecal bile acids (-45%) and a 3-fold increase in fecal cholesterol secretion. These effects were completely abolished in PPARalpha-null mice. A decreased bile acid production by PPARalpha-mediated downregulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase may contribute to the increased risk of gallstone formation after fibrate treatment.