The influence of HLA mismatches between donor and recipient on the phenotypes, function, and specificity of T-lymphocyte cultures derived from endomyocardial biopsies was studied in 118 heart transplant recipients. In case of HLA-DR mismatches, the majority of the EMB-derived cultures were dominated by CD4+ T cells while, in patients with HLA-A and -B mismatches but without DR mismatches, CD8+ T cells comprised the predominant T-cell subset. Cytotoxicity against donor antigens was observed in 75% of the cultures. A significantly (p < 0.005) lower proportion of the cultures showed cytotoxicity against HLA-A antigens (36%) when compared with HLA-B (53%) or HLA-DR (49%). An HLA-A2 mismatch elicited a cytotoxic response that was comparable to that found against HLA-B and -DR antigens: 62% of the cultures from HLA-A2 mismatched donor-recipient combinations was reactive against A2. A higher number of A, B, or DR mismatches resulted in a higher number of cytotoxic cultures directed against these antigens. A higher number of HLA-B and -DR mismatches was associated with a lower freedom from rejection. Our data indicate that, despite the use of adequate immunosuppressive therapy, the degree of HLA matching plays a crucial role in the immune response against a transplanted heart, resulting in a significant effect on freedom from rejection.