We have synthesized a series of N-phenyl-N'-aralkyl and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). This intracellular enzyme is thought to be responsible for the esterification of dietary cholesterol; hence inhibition of this enzyme could reduce diet-induced hypercholesterolemia. For this series of compounds, the in vitro ACAT inhibitory activity was improved by increasing the bulk of the 2,6-substituents on the phenyl ring. Additionally, we found that spacing of the aromatic rings was critical for ACAT inhibitory activity. A phenyl ring five atoms away from the requisite 2,6-diisopropylphenyl moiety was optimal for in vitro activity. Substitution alpha to the N'-phenyl moiety enhanced in vitro potency. In the case of phenylcycloalkyl ureas, ACAT inhibitory activity was independent of the size of the cycloalkyl ring. From this series of analogs, compound 25, which had excellent in vitro potency for inhibiting ACAT, was found to lower plasma cholesterol by 73% in vivo when administered in the diet at 50 mg/kg in an animal model of hypercholesterolemia. In this model, compound 25 lowered plasma cholesterol dose dependently and was as efficacious as the Lederle ACAT inhibitor CL 277082.