An adenoviral construct encoding a nuclear-localized β-galactosidase marker protein was injected into the heart of chick embryos at Hamburger-Hamilton (HH) stage 14–15 (approximately 52–56 h of incubation). Reporter gene expression was determined 48–54 h after injection. Efficient gene transfer into endothelial cells (ECs) of intraembryonic and yolk sac vessels was observed. ECs of vessels in the head region, which undergo massive expansion around the time of injection, were efficiently labeled. However, limb bud vasculature, which starts to develop around stage 16 (HH), carried scarce (wing bud) or no (leg bud) lacZ marker. In contrast, ECs of the allantois, a structure that develops even later (around stage HH 18), expressed lacZ reporter. This observation suggests that EC precursors infected at an earlier time migrated into the allantois. A few non-endothelial cell types were also labeled by the reporter. These results suggest that adenovirus-mediated gene transfer provides a powerful tool to study angiogenesis in the developing chick embryo.