Esterification of endogenous cholesterol in human small intestinal mucosa by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) was studied using [I-14C]oleoyl-CoA as substrate. The reaction was linear for 2 min only. The esterification of cholesterol was stimulated by albumin, but this effect was dependent on the oleoyl-CoA concentration. When the albumin concentration was 5 g/l, maximal esterification was obtained with 35 .mu.M oleoyl-CoA. The pH optimum was 7.2-7.8. The ACAT specific activity was highest in microsomal preparations from jejunum (0.21 .+-. 0.19 (n = 8) nmol cholesteryl oleate .cntdot. mg microsomal protein-1 .cntdot. min-1) and lower in proximal duodenum and distal ileum. Whole biopsy homogenates had about 1/4 of the activity of the corresponding microsomal preparation. Microsomal preparations from jejunum contained acyl-CoA hydrolase (EC 3.1.2.2), which under the prevailing conditions had a maximal activity of 4.4 nmol oleate formed .cntdot. microsomal protein-1 .cntdot. min-1. The high activity of intestinal ACAT in man renders it possible for this enzyme to play a role in cholesterol absorption.