Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome. The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown. An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses. The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2. Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro. The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR.