Adenovirus-mediated gene delivery is limited by the induction of immune responses that produce toxicity and prevent reexpression. To determine whether adenoviral delivery in the preimmune fetus would produce tolerance, we assessed luciferase (luc) expression following sequential pre- and postnatal adenoviral-mediated gene delivery. Day 15 fetuses were injected intrahepatically with 1 x 10(7) pfu of an adenoviral-luc vector (Ad-luc). Following in utero injection, hepatic luc expression persisted 1 month postnatally. No humoral response to adenovirus or luc was detected. Adult mice, previously injected in utero, were reinjected intravenously with 5 x 10(8) pfu of Ad-luc at 3 months of age and again at 6 months with either 5 x 10(8) pfu of Ad-luc or cationic liposome-DNA complexes (CLDC). Following the first postnatal injection, animals injected in utero had levels of luc comparable to those of age-matched naive controls. However, both control and experimental animals subsequently developed antibodies to adenovirus and luc. No further expression was achieved with a second postnatal injection of Ad-luc or with delivery of CLDC-luc. These studies demonstrate that the delivery of adenoviral vectors in utero at E15 does not elicit an immune response. However, delivery of recombinant adenovirus postnatally results in brisk and limiting immune responses regardless of the in utero exposure.