A substantial proportion of human peripheral lymphocytes exhibits esterase activity as demonstrated by cytochemical techniques using hexazotized pararosanilin as coupling agent and different alpha-naphthyl compounds, in particular alpha-naphthyl-acetate, as substrates. Esterase-positive lymphocytes showed one or several dots of the reaction product. Plasma cells and macrophages also exhibited marked esterase activity but with diffuse distribution of the reaction product throughout the cytoplasm. The substrate specificity, the time course and the pH optimum of the cytochemical reaction were determined. The results indicate that these lymphocyte esterases are identical with acid lipases. The number of esterase-positive cells reaches a plateau well below 100% (85% for blood lymphocytes) even with optimal staining procedures. The relation between esterase activity and other so-called lymphocyte markers such as sIg and the capacity to form spontaneous rosettes with sheep red blood cells was investigated in peripheral human blood lymphocytes. A fair correlation between rosette formation and esterase activity was found and most of the esterase-positive cells were sIg-negative. However, the fit was never complete. The results suggest that esterase activity is largely characteristic for small peripheral T cells. Almost all blast cells formed following stimulation with Con A or PHA were esterase-positive. Histochemical studies showed that the large majority of the thymocytes in the cortex lacked esterase activity. In lymph nodes, a low proportion of esterase-positive cells was found in the germinal centers, whereas most lymphocytes in the paracortical are were esterase-positive.