We show that replication defective adenovirus can be used for localized overexpression of a chosen gene in Xenopus tadpoles. Xenopus contains two homologs of the Coxsackie and Adenovirus Receptor (xCAR1 and 2), both of which can confer sensitivity for adenovirus infection. xCAR1 mRNA is present from the late gastrula stage and xCAR2 throughout development, both being widely expressed in the embryo and tadpole. Consistent with the expression of the receptors, adenovirus will infect a wide range of Xenopus tissues cultured in vitro. It will also infect early embryos when injected into the blastocoel or archenteron cavities. Furthermore, adenovirus can be delivered by localized injection to tadpoles and will infect a patch of cells around the injection site. The expression of green fluorescent protein in infected cells persists for several weeks. This new gene delivery method complements the others that are already available. Developmental Dynamics 238:1412–1421, 2009. © 2009 Wiley-Liss, Inc.