Most known DNA replication origins in vertebrate genomes have been found to occur close to transcriptional promoters. The origin of bidirectional DNA replication of the chicken lysozyme GAS41 locus was identified in a CpG island covering the GAS41 gene promoter. In this study, we generated an α-Orc2 antibody from rabbits immunized with the C-terminal half of Orc2 for studying in vivo Orc2 binding to the lysozyme-GAS41 origin. Using the chromatin immunoprecipitation technique and quantitative real-time polymerase chain reaction, we were able to map the Orc2-binding site to a region of the lysozyme GAS41 origin that contains multiple Sp1/Sp3-binding sites co-mapping with two DNase I hypersensitive sites. Further, knockdown of endogenous Sp1 by RNA interference reduced specific Orc2 binding to the lysozyme GAS41 origin. These results suggest that Sp1 participates in recruiting Orc2 to the origin.