Acyl-Coenzyme A:cholesterol Acyltransferases (ACATs), members of the membrane bound O-acyltransferase (MBOAT) family, catalyze the conversion of cholesterol to cholesteryl esters. Mammals contain two isoenzymes ACAT1 and ACAT2. Both enzymes are drug targets for treating human diseases. ACAT1 is present ubiquitously in various cell types. It contains 9 transmembrane domains (TMDs) with the active site H460 locating within TMD #7, and another active site N421 locating within the 4th large cytoplasmic loop. In human ACAT1, a single nucleotide polymorphism (SNP) exists for residue 526: the codon is either CAG for glutamine (Q), or CGG for arginine (R). Q/R 526 is present within the C-terminal loop. Its biochemical significance is unknown. In addition, within the C-terminal half of ACAT1, numerous residues conserved with those of ACAT2 are present; the functions of these conserved residues are largely unknown. Here we performed single substitution mutagenesis experiments to investigate the roles of individual residues present in the C-terminal loop including Q/R526, and the 8 conserved prolines (P) located near/at various TMDs. The results show that the enzyme activity of ACAT1 Q526 is less active than that of ACAT1 R526 by 40%. In addition, several residues in the C-terminal loop are important to maintain proper ACAT1 protein stability. Other results show that the P347 plays important role in modulating enzyme catalysis. Overall, our results implicate that the CAG/CGG polymorphism can be utilized to perform ACAT1 activity/human disease susceptibility studies, and that P347 located near TMD #5 plays an important role in modulating enzyme catalysis.