Rat lymphocyte populations were assessed for 5'-nucleotidase activity by both biochemical and histochemical methods. Enzyme-specific activity was enriched in lymphocyte plasma membrane fractions and was higher in lymph node and spleen lymphocytes than in thymocytes. Histochemical reactions on sections of spleen and lymph node revealed strong activity in the thymus-dependent regions, periarteriolar lymphoid sheath in spleen, and paracortex in lymph node; whereas the thymus-independent regions, follicles, and germinal centers were negative. In cellular depletion experiments, with three different methods to detect 5'-nucleotidase, it was observed that the depletion of T cells, but not B cells, was accompanied by a loss of enzyme activity and a decrease in the percentage of nucleotidase-positive cells. The results suggest that, among members of the lymphocyte series, high 5'-nucleotidase activity is selectively associated with the plasma membranes of peripheral T cells in the rat.