Nucleotide sequences of the intergenic region between ORF 62 and ORF 63 of equine herpesvirus 1 (EHV-1) isolates were analyzed. The sequences of this region consisted of variable and conserved domains among EHV-1 isolates. An EHV-1 mutant, Ab4-GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by loxP at both ends into the intergenic region between ORF 62 and ORF 63. Another mutant, Ab4-loxP, which contains one loxP site, was constructed by excision of the GFP cassette from the Ab4-GFP virus genome by cre enzyme. The recombinant Ab4-GFP formed smaller plaques than the wild type in MDBK cells. Virus production also decreased for Ab4-GFP in multi-step growth analyses. Virulence of Ab4-GFP in both mice and hamsters was weaker than that of the wild type. Ab4-loxP exhibited properties similar to those of the wild type. These results suggest that the intergenic region between ORF 62 and ORF 63 plays various roles in the virus growth.