Cell surface markers and cytochemical characteristics of cynomolgus lymphocytes were studied. The proportion of lymphocytes with spot-like distribution of acid alpha-naphthyl acetate esterase (ANAE) activity was similar to that of lymphocytes which form spontaneous rosette with sheep erythrocytes (E rosette) in individual monkeys. Fractionation by E rosette formation or passage through nylon wool column selectively enriched ANAE-spot-positive (ANAE+) lymphocytes and resulted in twofold increase in blastogenic response when stimulated with phytohemagglutinin (PHA) or concanavalin A (Con A). In contrast, treatment of lymphocytes with rabbit anticynomolgus thymocyte serum (ATS) plus complement resulted not only in the depletion of both E rosette forming and ANAE+ lymphocytes, but also in the reduction of lymphocyte blastogenic response to PHA or Con A. In cryostat sections of lymphoid tissues. ANAE+ lymphocytes were found to reside in T dependent areas such as paracortical areas and periarterial lymphatic sheaths but not in T independent areas. ANAE activity was absent, however, in cortical thymocytes and lymphoblasts transformed by phytomitogens, suggesting that immature T lymphocytes are ANAE negative. These findings indicate that the histochemical demonstration of ANAE activity and E rosette formation serve useful markers for T lymphocytes in cynomolgus monkeys as in human.