The capacity of E1A gene-deleted and thus replication-defective adenovirus type 5 (Ad5) to transduce foreign genes in chicken embryo fibroblasts (CEF) as well as in chickens was investigated. The lacZ and luciferase genes were successfully transduced in CEF by replication-defective Ad5, demonstrating that these cells possess receptor(s) for binding and penetration of Ad5. A single intramuscular inoculation of Ad-gD, a replication-defective Ad5 harbouring the gD gene of pseudorabies virus, in adult and 1-day-old chickens led to the production of very high titres of specific antibodies. These gD-specific antibodies persisted for at least 56 days. These results demonstrate that replication-defective Ad5, despite its mammalian origin and the deletion of the E1A gene, is a good candidate for developing non-spreading vaccines in poultry.