The role of cytotoxic cells in in vivo immune functions such as allograft rejection is unknown. To begin to assess the function of cytolytic cells in vivo we have begun with cytolytic cell-specific functional molecules: we have isolated and characterized cytolytic cell-specific cDNA clones from cytolytic T cell clones, both encoding distinct serine esterases. The HF gene encodes a trypsin-like enzyme while the C11 gene encodes an enzyme with likely specificity for acidic residues. Here we demonstrate, using in situ hybridization with RNA probe, that both genes are expressed selectively in a subset of T lymphocytes that have infiltrated cardiac allografts. The phenotype of these cells is consistent with the most frequent phenotype of active CTL raised in vitro: they are predominantly CD4-, CD8+, MEL-14- T cell blasts. Thus the expression of these genes, each of which encodes serine esterase found in killer cell granules in vitro, is a valid marker for these cells in vivo as well. The kinetics of their accumulation is consistent with, but not proof of, a putative role in allograft rejection. It is likely that HF and C11 gene expression will be of diagnostic value.