In order to further define the structural features necessary for potent inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in vitro and cholesterol lowering in vivo, systematic study of bioisosteric replacements for the amide bond in our previously identified series of fatty acid anilide ACAT inhibitors was undertaken. Only replacement of amide bonds with isosterases having both hydrogen bond donor and acceptor functionalities yielded compounds retaining ACAT inhibitory activity. Replacement of the amide bond with the urea bioisostere yielded compounds that were potent ACAT inhibitors in vitro and efficacious hypocholesterolemic agents in vivo. Examination of the structure activity relationships in the phenyl ring and alkyl portion of the N-phenyl-N'-alkylureas revealed that 2,6-diisopropyl substitution was optimal in the phenyl ring. When the 2,6-diisopropyl moiety was kept constant, potency in vitro and in vivo was maintained with straight and branched alkyl groups from 6 to 18 carbons in length.