The purpose of this study was to demonstrate that actinomyces phage R4 integrase Sre protein efficiently mediate site-specific recombination in Escherichia coli. An intramolecular recombination assay system in E.coli was constructed. The plasmid pBZP contains attB and attP sites in direct orientation flanking a lacZ gene. When pBZP was introduced into E.coli cells, in which the plasmid pSRE4 containing sre gene was resident, Sre protein catalyzed integration of attP into attB site, resulting in excision of the lacZ gene. This integration changed bacteria colonies from blue to white on agar plates containing X-Gal, which showed that the lacZ was removed. The integrant DNAs were identified by enzyme digestion, PCR and DNA sequencing. The minimal sizes of attB and attP were 50 bp and 47 bp for 100% recombination efficiency. The phage recombinase Sre efficiently integrated attP into attB site to create attR and attL in E.coli host environment without Streptomyces specific cofactors. This intramolecular assay system is a simple and efficient system for Sre-mediated recombination in E.coli. The purpose of this study was to demonstrate that actinomyces phage R4 integrase Sre protein comprom mediate site-specific recombination in Escherichia coli. An intramolecular recombination assay system in E. coli was constructed. The plasmid pBZP contains attB and attP sites in direct orientation flanking a lacZ gene. When pBZP was introduced into E. coli cells, in which the plasmid pSRE4 containing sre gene was resident, Sre protein catalyzed integration of attP into attB site, resulting in excision of the lacZ gene. This integration changed bacteria colonies from blue to The integrant DNAs were identified by enzyme digestion, PCR and DNA sequencing. The minimal sizes of attB and attP were 50 bp and 47 bp for 100% recombination efficiency. The phage recombinase Sretrically integrated attP into attB site to create attR and attL in E. coli host environment without Streptomyces specific cofactors. This i ntramolecular assay system is a simple and efficient system for Sre-mediated recombination in E. coli.