A new substrate optimized assay for acyl-CoA:cholesterol acyltransferase (ACAT) was developed that permits the accurate measurement of ACAT activity in normal arterial microsomes. The apparent Km and Vmax of ACAT with respect to oleoyl-CoA were determined to be 3 microM and 17.7 pmole min-1 mg-1. While the Km value is similar to other values reported in the literature, the Vmax is 5- to 8-fold higher. The higher Vmax is attributable to the saturation of ACAT with not only oleoyl-CoA, but also cholesterol. The observation that exogenous cholesterol was necessary for the determination of maximal ACAT activity indicates that under normal conditions the endogenous level of microsomal cholesterol does not saturate ACAT. Assay of ACAT in the presence and absence of exogenous cholesterol permits a qualitative assessment of the amount of cholesterol in the cholesterol substrate pool of ACAT. Using this approach, it was found that hypercholesterolemia results in the expansion of the cholesterol substrate pool of ACAT. Of the 21-fold increase in ACAT activity in atherosclerotic aortas observed in this study. 80% of the increase was attributable to expansion of the cholesterol substrate pool, while 20% was attributable to more enzyme. Notably, the increase in the amount of ACAT was observed after only 2 weeks of hypercholesterolemia.