Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-complex peroxidase technique, formalin-fixed, paraffin-embedded tissue sections from patients with acute mild or moderate allograft cardiac rejection were stained. A constant cytoplasmic staining of the lymphocytic allograft infiltrate was observed. These results provide a basis for using the anti-granzyme B antibodies for detection of cytotoxic cells in human tissues. The detection and quantitative analysis of the granzyme-associated cytotoxic cells may help to evaluate the significance of these functionally distinct cytotoxic cells in human tissues associated with increased expression of cytoplasmic granule effector molecules.