T lymphocytes used for adoptive immunotherapy are often cultured before transfer to generate sufficient amounts of effector cells with desired specificity. Modification of lymphocytes induced by in vitro activation and expansion may influence their potential effector capacity by altering the survival and trafficking patterns after transfer. In this report, the authors show that the culture period of T cells after ConA/IL-2 stimulation strongly influences the retention and tissue distribution of these cells after infusion into syngeneic C57BL/6 mice. Infused labeled cells that have been cultured for 3 days remained in the peripheral blood and organs in at least a ten-fold higher number than cells cultured for 8 days. In addition, cells cultured for 3 days preferentially migrate to lungs and liver shortly after infusion and subsequently to lymph nodes and spleen. Cells cultured for 8 days preferentially migrate to liver and can be hardly detected in lymph nodes. In contrast, labeled cells cultured for 3 days are predominantly present in lymph nodes starting from day 8 until day 28. We showed that accurate monitoring of transferred cells is feasible, which may contribute to understanding response to adoptive immunotherapy.