As a possible explanation for everolimus/cyclosporine-induced hypercholesterolemia seen in transplant recipients, we investigated the interactions of the immunosuppressants everolimus and cyclosporine on bile flow and biliary excretion of bile salts and cholesterol in a subchronic bile fistula model in rats because biliary excretion is a main elimination route of cholesterol. After 2 weeks of daily treatment, everolimus (1 mg/kg i.p.) and cyclosporine (5 mg/kg i.p) decreased bile flow (-45 and -36%) and biliary excretion of bile salts (-34 and -54%) and cholesterol (-25 and -39%) and increased serum concentrations of cholesterol (+40 and +17%) and triglycerides (+220 and +110%). Bile salt serum concentration was elevated only by cyclosporine (+100%), and not by everolimus. Everolimus/cyclosporine slightly enforced the cyclosporine-induced hyperlipidemia but not reduction of bile parameters, while the cyclosporine-induced increase in bile salts in serum was totally prevented. From these results we conclude that bile salt synthesis could be impaired by everolimus, which could be one reason for everolimus-induced hypercholesterolemia.