Fatty acyl CoA and cholesterol are the substrates for cholesteryl ester synthesis by acyl coenzyme A:cholesterol acyltransferase (ACAT). Two ACAT genes have been identified; ACAT1 is expressed ubiquitously while ACAT2 is primarily expressed in intestine and liver. We tested effects of different free fatty acids (FFAs) on ACAT1 and ACAT2 expression and activity in HepG2 human hepatocytes and THP1 human macrophages. Incubation of oleic acid, arachidonic acid, or eicosapentaenoic acid, but not 25-hydroxycholesterol, induced ACAT1 mRNA levels 1.5--2-fold in HepG2, with no affect on ACAT2 mRNA. FFA had no affect on ACAT1 mRNA in THP1 cells. To determine if FFAs affect ACAT1 or ACAT2 posttranscriptionally, cells were labeled with [(3)H]cholesterol in the presence of the different FFAs for 1--5 h. Both HepG2 and THP1 cells showed the greatest cholesteryl ester production with oleic acid. This was also confirmed by the observation that more [(3)H]oleic acid incorporated into CE compared to [(3)H]eicosapentaenoic acid, even though there was no difference in the total uptake of these FFAs. In ACAT-deficient SRD4, CHO cells stably transfected with human ACAT1 or ACAT2, ACAT1 expressing cells showed a strong preference for oleic acid while ACAT2 expressing cells utilized unsaturated FFAs. Acyl CoA substrate specificity was further tested in microsomes isolated from these cells as well as HepG2 and THP1. THP1 and ACAT1 cells utilized oleoyl CoA preferentially. In contrast, HepG2 and ACAT2 microsomes utilized linolenoyl CoA as well. We conclude that FFAs increase ACAT1 mRNA levels in a cell specific manner, and furthermore that the ACAT reactions exhibit differential FFA utilization.