From 535 endomyocardial biopsies (87 heart transplant recipients) 283 cell cultures could be generated. All cultures tested contained T lymphocytes and in most cases CD4 was the predominant phenotype at any time posttransplant. A significantly higher proportion of CD8-dominated cultures was found among cultures from biopsies without myocytolysis. In the first 3 months post transplant 57% of cultures showed cytotoxicity against both class I and class II mismatched donor major histocompatibility complex (MHC) antigens, changing to an incidence of 33% at greater than 90 days. This proved to be due to a significant decrease in the number of cultures with human leukocyte antigen class II-directed cytotoxicity. This study shows that early after transplantation a heart transplant is infiltrated with activated donor-specific cytotoxic T cells which recognize a broad spectrum of mismatched donor MHC antigens, and that in time this spectrum becomes more restricted.