We previously showed that activated T cells can be propagated from EMB samples using recombinant IL-2 (rIL-2) and that a strong correlation exists between ability to culture T cells and histologic grade of rejection. Lymphocytes could not, however, be grown from many EMB even in the presence of rejection. We hypothesised that this may in part reflect the restrictive nature of the culture (rIL-2 as the only `growth factor') and that the derived cultures may not be fully representative of activated T cells within the graft. Methods: We therefore prospectively studied 116 consecutive EMB samples from 59 transplant recipients maintained on cyclosporine or tacrolimus (FK-506) based immunosuppression. Each was divided into multiple fragments and half were cultured in media containing 30U/ml rIL-2 with autologous irradiated peripheral lymphocytes as “feeder cells”. The remaining half were cultured in the same environment with the addition of 200U/ml rIL-4. Lymphocytic growth within 14 days was correlated with histological grade of rejection and culture environment. Phenotype analysis of cultures (CD4+/ CD8+) was performed by flow cytometry. Functional characterization (donor specificity) was assessed by primed lymphocyte test(PLT) using donor spleen as antigen. Results: Lymphocyte growth occurred in 10/93 (14%) grade 0/1a EMB & 10/23 (43%) grade ≥ 1b. Of the 20 positive cultures, growth was greatest in media containing rIL2 + rIL4 in 12 (including 6 with no growth in rIL-2 alone), in rIL-2 alone in 2, and comparable in the two environments in 6. Flow cytometry revealed no significant difference in phenotype between culture environments and PLT showed comparable proliferative responses to donor antigen.Conclusions: Positive lymphocyte growth is strongly associated with higher grade of rejection. The addition of rIL-4 enhances propogation of donor specific T cells from EMB, thus providing more material for functional studies of graft infiltrating cells. No phenotypic or functional differences between cells cultured in the 2 environments have been demonstrated, but it is postulated that cytokine analysis of proliferating cultures might reveal functional heterogeneity within CD4+ or CD8+ populations.