In vivo activated T-lymphocytes can be cultured from endomyocardial biopsy samples of human cardiac allografts, sometimes even in the absence of histological rejection. We investigated the clinical relevance of this "lymphocyte growth assay" in pediatric heart transplant recipients. Specifically, we wished to determine if: (i) positive lymphocyte growth from EMB samples in the absence of significant rejection identifies a patient as being at increased risk for the development of acute rejection; (ii) withdrawal or major dose reduction of corticosteroids in the presence of lymphocyte growth results in high risk of rebound rejection; and (iii) presence of lymphocyte growth during acute rejection helps predict the response to treatment. Cultures were performed on 789 consecutive EMB samples from 65 pediatric heart transplant recipients in media containing 30 U/ml of recombinant IL-2. T-lymphocytes were cultured from 16% of EMB samples with low grade rejection (grade 0-1b) and from 34% of EMB samples with grade 2-4 rejection. EMB samples obtained early post-transplant (<180 days) were significantly more likely to yield positive lymphocyte growth compared to biopsies obtained late for any given rejection grade. Lymphocyte growth was comparable between patients managed with cyclosporine or tacrolimus based immunosuppression. For 227 EMB samples without rejection, a subsequent EMB sample was obtained within 12 weeks. Lymphocyte cultures were positive in 47 of these 227 EMB samples (21%), and in 19 out of 47 (40%) cases acute rejection (grade 2-4) was present on the follow-up EMB sample. By contrast, of 180 biopsies without growth, only 29 (16%) showed rejection at the next EMB (p<0.0001). When a follow-up biopsy was performed within 12 weeks of corticosteroid withdrawal, "rebound rejection" was observed in 3 out of 10 (30%) cases where the previous EMB sample yielded positive lymphocyte growth and in 4 out of 38 (11%) cases when it did not (p=0.29). The presence of lymphocyte growth in association with rejection was also predictive of whether rejection would resolve following high dose intravenous corticosteroid therapy (persistent rejection in 33 out of 50 (66%) cases with positive growth, versus 25 out of 80 (31%) cases without growth (p<0.0001)). Thus, positive lymphocyte growth is strongly associated with higher grade of rejection and earlier time from transplantation. Lymphocyte growth in the absence of rejection indicates high risk for rejection within the next 12 weeks. Growth in association with acute rejection indicates high probability of persistence of rejection following treatment with high dose corticosteroids.