Specific esterase isoenzyme patterns in plasma may be associated with responsiveness of serum cholesterol to dietary cholesterol. In rabbits and rats the presence and absence of a high-mobility, anodal esterase band on electrophoresis have been shown to be associated with hypo- and hyperresponsiveness, respectively. We fed for 28 days male mice of 7 inbred strains either a low-cholesterol, commercial diet or a diet containing 2% (w/w) cholesterol, 0.5% cholic acid and 5% olive oil. Feeding the high-cholesterol diet revealed marked inter-strain differences in the responses of plasma and liver cholesterol; the increases ranged from 21 to 129% and from 10 to 80-fold, respectively. There was no association between esterase isoenzyme patterns in plasma and the sensitivity to the high-cholesterol diet. The mean baseline plasma total esterase activity tended to be positively associated with the absolute response of plasma cholesterol to the high-cholesterol diet (r = 0.56; n = 7), but the positive relationship between the baseline concentration of the ES-1 component in plasma and the cholesterolemic response was stronger (r = 0.84; n = 7; P less than 0.05). The high-cholesterol diet caused a significant increase in plasma total esterase activities in 6 out of the 7 strains. Evidence is presented that the increase in plasma total esterase activity, which was associated with an increase in the activity and concentration of the so-called ES-2 isoenzyme, is the result of an enhanced release of esterases from the intestine, rather than from the liver. A significant, positive correlation was found between the baseline intestinal esterase activity and the cholesterolemic response after cholesterol feeding (r = 0.83; n = 7; P less than 0.05).