1. The submicrosomal distribution of three enzymes concerned in cholesterol metabolism, and of free and esterified cholesterol, was determined in human liver by analytical isopycnic centrifugation on sucrose gradients.

2. The distribution profile and median density of acyl-CoA:cholesterol O-acyltransferase was similar to that of RNA, showing that this enzyme is confined largely to the ribosome-rich membranes of the endoplasmic reticulum. The distribution profiles and median densities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7α-mono-oxygenase showed that both enzymes are confined to the smooth, ribosome-poor, endoplasmic reticulum.

3. Most of the free cholesterol in the microsomal preparations was present in smooth membranes from the Golgi apparatus and in vesicles from plasma-membrane fragments. The distribution of esterified cholesterol was multimodal and extended throughout the whole gradient.

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