Aging is associated with alterations in the functions of the intestine. Also, the functions of the intestine vary along the length of the villus. A method is reported for the isolation of four fractions of enterocytes from along the villus of young (3 months, 300-350 g) and old (8 months, 700-750 g) male Wistar rats. Different qualitative patterns are seen when the activities of alkaline phosphatase (AP), invertase (INV), or [3H]methylthymidine incorporation are expressed on the basis of millimoles per gram protein, percentage of highest activity, percentage of total activity, cumulative activity for each fraction, or millimoles per gram protein versus cumulative percentage of total protein. When expressed on the basis of protein, AP is higher in upper (FI) versus lower (FIV) villus fractions, and in each fraction AP is higher in old versus young animals. INV varies little along the villus or between young and old rats; [3H]methylthymidine incorporation is highest in the crypt cells. The ratio mg protein/mg DNA varies along the villus and is twofold higher in young than in old rats. In contrast, when activities are expressed on the basis of DNA, the same qualitative patterns are seen along the villus, but INV and AP are both higher in young than in old rats. In summary (i) the qualitative and quantitative differences in the activities of AP and INV along the villus of old versus young rats vary when the data are expressed on the basis of DNA rather than protein; (ii) the ratio of protein to DNA varies along the villus in young and old animals, and for this reason it is appropriate to report enzyme activity on the basis of enterocyte DNA; and (iii) INV and AP, when expressed on the basis of DNA, are higher in young than in old animals. Thus, the isolation of rat jejunal enterocytes from along the villus is affected by the age of the animals.