A modified technique was used to demonstrate lymphocytic acid α-napth acetate esterase activity in frozen sections of mouse lymphoid tissue or smears. Positive, dot-like reaction products were noticed in more than 94 % of the lymphocytes located in the diffuse cortical (“paracortical”, “thymus-dependent”) area of mesenteric lymph nodes of young adult ICR mice. Almost identical values were found for lymphocytes in the cisterna chyli. In contrast, the follicular cortex, which is predominantly occupied by B cells, contained less than 7 esterase-positive lymphocytes. In vitro, cytotoxic anti-θ serum destroyed vast majority of esterase-positive lymphocytes while esterase-negative lymphocytes were resistent to this treatment. In mesenteric nodes of nude BALB/(nu/nu) mice, follicular cortex and paracortex together contained approximately 16 times less esterase-positive lymphocytes than in the immunologically competen hybrid BALB/c (nu/+) animals. These findings indicate that non-specific acid esterase activity may serve as a criterion to differentiate peripheral T B lymphocytes in lymphocytes in lymph node sections and smears of mice by light microscopy Possible implications of this enzyme activity in thymus-derived lymphocytes are discussed.