Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular mass; dot blot hybridization with synthetic oligonucleotides corresponding to the active sites of two known murine CTL esterases suggests homology to the murine enzyme HF. However, serine esterase was present at only approximately 10% of the level found in murine CTLs, and was not secreted during CTL-target cell interaction; moreover, hemolytic activity could not be detected in any of the seven cell lines tested. The results suggest that the human CTLs examined here kill their target cells by a mechanism different from that used by most cloned murine CTLs.