Cytotoxic T lymphocytes and lymphocytes with NK-like activity contain a serine esterase activity which has been localized to their cytoplasmic granules by cytochemistry and subcellular fractionation studies. The serine esterase-specific inhibitor 3H-DFP labels two protein species in the granules. The two proteins, referred to as serine esterases 1 and 2 (SE 1 and SE 2), migrate with Mr of 34-36 kd and 28-30 kd, respectively, under reducing conditions. SE 1 shows trypsin-like activity and has been purified to apparent homogeneity. Under nonreducing conditions, SE 1 has an Mr of 60-66 kd, suggesting that it may consist of two disulfide-linked subunits of 34-36 kd each. SE 1 cleaves fibrin and casein, has a pl greater than 10, and optimal activity at pH 8. The substrate specificity of SE 2 is not known. The serine esterase activity is secreted by lymphocytes that have been stimulated with the calcium ionophore A23187. The serine esterases described here could play an active role in cell-mediated killing.