Blockade of the CD40-CD40L and CD80/CD86-CD28 costimulatory pathways represents a strategy to inhibit the immune response against Ad vectors designed for gene therapy applications. Since most previous studies have used a CTLA4-Ig fusion molecule binding to both CD80 and CD86, the respective roles of these B7 molecules remained undefined. We have studied the effect of blocking monoclonal Abs (mAbs) directed against the costimulatory molecules CD40L, CD80 and CD86, alone or in different combinations, on the humoral and cellular immune responses against Ad. Groups of mice were transiently treated with each combination of blocking mAbs upon systemic injection of a first Ad vector. Combinations of anti-CD80 + anti-CD86 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition of the immune response against Ad. Using either of these mAb pairs, a second vector could be administered 1 month after the first injection but with lower efficiency than in naive animals. Thus, CD86 stands as the pivotal B7 molecule involved in the development of the immune response against Ad. However, only the blockade of both CD80 and CD86 in addition to CD40L fully inhibited the humoral and cellular responses against the Ad vector, such that readministration after 1 month was as efficient as in naive animals. At the time of readministration, treated animals had regained their ability to mount a normal immune response to the second Ad vector, showing that tolerance was not induced.