A series of N-alkyl-N-biphenylylmethyl-N′-arylurea and related derivatives represented by 1 have been prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O-acyltransferase in vitro and to lower plasma cholesterol levels in cholesterol-fed rats in vivo. Linking of two phenyl groups via oxygen and introduction of fluorine at appropriate positions on the biphenyl moiety improved in vitro and in vivo activity. From this series of analogs, compound 40 (FR179254), which had potent in vitro potency (rabbit intestinal microsomes IC₅₀ = 25 nM), showed excellent plasma cholesterol-lowering activity when administered via the diet (ED₅₀ = 0.045 mg/kg). However, the hypocholesterolemic effect of this compound was moderate when dosed by oral gavage in PEG400 as a vehicle (ED₅₀ = 5.3 mg/kg). Modification of the N′-aryl moiety led to the identification of compound 50 (FR182980) which was efficicious in both dosing models (ED₅₀ = 0.034 mg/kg and 0.11 mg/kg, respectively.