Abstract The transfer of non-esterified cholesterol to rat-liver microsomal fraction resulted in a considerable decrease in the activity of 5′-nucleotidase and in changes in the characteristics of the Arrhenius plots of the enzyme. The decrease in the activity of 5′-nucleotidase and the increase in the concentration of non-esterified cholesterol in the serum-treated preparations were serum-concentration-dependent and incubation-time-dependent. The enzyme in serum-treated preparations with high non-esterified cholesterol content showed Arrhenius plots with a constant activation energy between 37 and 19°C, whereas the enzyme in the non-treated microsomal fraction or the lipoprotein-deficient serum-treated preparations showed a break at about 28°C, with activation energies higher below and lower above the break. These changes in the temperature-induced kinetics are consistent with an increase in the concentration of non-esterified cholesterol in the plasma membrane vesicles of the serum-treated preparations. The Arrhenius plots of 5′-nucleotidase in liver microsomal fraction from rats fed cholesterol-supplemented diet showed constant activation energy between 37 and 19°C and had similar characteristics with the plots for 5′-nucleotidase in serum-treated preparations. Since the changes in the characteristics of Arrhenius plots of the enzyme in microsomal fraction from rats that had been denied food for 36 h were in the opposite direction to those produced by feeding cholesterol, these results are consistent with a lower concentration of non-esterified cholesterol in hepatic plasma membranes from fasted rats relative to that in plasma membranes from fed rats. The isolation of a plasma membrane preparation with negligible contamination of endoplasmic reticular membranes from rats fed the standard or cholesterol-supplemented diet and from fasted rats showed that the ratio of cholesterol to phospholipid has increased in the preparation from rats fed cholesterol and decreased in that from rats that had been denied food relative to the ratio in the preparation from rats fed the standard diet. The Arrhenius plots of 5′-nucleotidase in these preparations showed characteristics similar to the corresponding plots of the enzyme in the microsomal fraction from the rats in the three experimental conditions.