We have examined the kinetics of [14C]cholesterol exchange between unilamellar vesicles formed from the following synthetic glycerophosphatidylcholines: (a) those having acyl (OC(O)R), acylamino (NHC(O)R), carbamoyl (NHC(O)OR), and acylthio (SC(O)R) chains at the sn-2 position, and (b) those having alkyl (OR) and thioalkyl (SR) chains at the sn-1 position. Replacement of the glycerol oxygen atom at the sn-2 position of PC with a NH group did not affect the rate of cholesterol exchange to a significant extent, suggesting that the amide group of sphingomyelin is not primarily responsible for the very slow rate of exchange of cholesterol observed from sphingomyelin vesicles. Replacement of the glycerol oxygen at the sn-2 position of phosphatidylcholine with a sulfur atom caused the rate of spontaneous cholesterol exchange to increase by a factor of 1.6. Substitution of an O-alkyl chain for the acyl chain at the sn-1 position of 2-acylthiophosphatidylcholine or substitution of a thioalkyl chain for the O-alkyl sn-1 chain of 1-alkyl-2-acylaminodeoxyphosphatidylcholine also did not result in a marked difference in cholesterol exchange rate. The data suggest that interactions other than intermolecular hydrogen bonding are involved in determining the rates of intermembrane cholesterol exchange. Significantly, these kinetic studies also lend support to the continued use in model membranes of synthetic sulfur- and nitrogen-substituted phosphatidylcholines, which have been employed to study properties of lipolytic enzymes, since synthetic acylamino- and acylthio-phospholipids form vesicles that give cholesterol exchange rates that closely resemble those found in vesicles prepared with diester-phosphatidylcholines.