Ad-ISF35 is an adenovirus (Ad) vector that encodes a mouse-human chimeric CD154. Ad-ISF35 induces activation of chronic lymphocytic leukemia (CLL) cells converting them into CLL cells capable of promoting immune recognition and anti-leukemia T-cell activation. Clinical trials in humans treated with Ad-ISF35-transduced leukemia cells or intranodal injection of Ad-ISF35 have shown objective clinical responses. To better understand the biology of Ad-ISF35 and to contribute to its clinical development, we preformed studies to evaluate biodistribution, persistence and toxicity of repeat dose intratumoral administration of Ad-ISF35 in a mouse model. Ad-ISF35 intratumoral administration induced tumor regression in more than 80% of mice bearing A20 tumors. There were no abnormalities in the serum chemistry. Mice receiving Ad-ISF35 presented severe extramedullary hematopoiesis and follicular hyperplasia in the spleen and extramedullary hematopoiesis with lymphoid hyperplasia in lymph nodes. After Ad-ISF35 injection, the vector was found primarily in the injected tumors with a biodistribution pattern that showed a rapid clearance with no evidence of Ad-ISF35 accumulation or persistence in the injected tumor or peripheral organs. Furthermore, pre-existing antibodies against Ad-5 did not abrogate Ad-ISF35 anti-tumor activity. In conclusion, intratumoral administration of Ad-ISF35 induced tumor regression in A20 tumor bearing mice without toxicities and with no evidence of vector accumulation or persistence.