Graft-infiltrating cells can be cultured from fresh endomyocardial biopsies (EMB) taken after heart transplantation to determine their growth patterns, phenotypic composition, and functional characteristics for clinical or scientific purposes. In this study we investigated whether graft-infiltrating cells can also be cultured successfully after cryopreservation of these EMB. Three different cryopreservation methods were used. One method gave successful growth in 100% of the cases (n = 6): The biopsy fragments were preincubated in 10% vol/vol dimethyl sulfoxide during 5 min at 0 degree C, frozen to -70 degrees C at approximately 1 degree C per minute, and subsequently immersed and stored in liquid nitrogen. Thawing was performed rapidly in water at 37 degrees C. In addition, the effect of cryopreservation on cell surface phenotype and donor-specific cytotoxicity of these graft-infiltrating cells was analyzed. When compared to cultures of nonfrozen control biopsies, both qualities remained constant in most cases, although a variation in CD4+/CD8+ cell ratio was observed in 33% of these cultures. However, when nonfrozen fragments of size-matched biopsies were cultured separately, a similar variation in phenotype was noted, indicating that this phenomenon can be attributed to sampling variation and not to the cryopreservation procedure. The present findings suggest that it is no longer required to culture fresh (nonfrozen) post-transplant EMB to propagate graft-infiltrating cells: Culturing can be limited to cryopreserved EMB that are selected retrospectively, depending on actual clinical or scientific interests. Besides greatly facilitating the long-term monitoring of heart transplant recipients, this also means a substantial decrease in cost and work load for laboratories involved in heart transplantation.