Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC₅₀ values of 100 and 1,000 μM, respectively, in the tubules. Next, the inhibitor concentration (100 μM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 μg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 μg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0–1,000 μM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.