The use of granzyme A and perforin as markers for rejection after cardiac transplantation has been investigated. Using in situ hybridization we have detected lymphocytes expressing granzyme A and perforin RNA that are infiltrating the donor heart after transplantation. A total of 29 different biopsies from 17 different patients who had undergone cardiac transplantation were examined. Twelve biopsies classified by conventional histological criteria as showing evidence of rejection were found to contain lymphocytes expressing granzyme A and perforin. Seven biopsies classified as showing no histological evidence of rejection infiltrating lymphocytes were found not to be expressing granzyme A or perforin. However, in 10 other biopsies from 5 different patients that had been classified as showing no evidence of rejection by the conventional grading system, lymphocytes expressing granzyme A and perforin were detected. In six of these cases the patient was found to have undergone a subsequent rejection episode. In the other four cases the biopsies were either taken at a very early stage after transplantation and the high doses of immunosuppression used routinely at that stage are likely to have averted any rejection episodes, or it was not possible to follow subsequent rejection episodes. These results, which are statistically significant (p = 0.06), demonstrate that granzyme A- and perforin-expressing lymphocytes can be identified in rejecting biopsies before histological damage is seen. The identification of perforin and granzyme A expression in vivo suggest a possible role for these proteins in the cytolysis that occurs during transplantation rejection. Furthermore, the data presented here suggest that it may be possible to use granzyme A and perforin as early predictive markers of transplantation rejection.