This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage attsites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage attsite with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attBand attPsites at a frequency of ∼ 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attBand attPsites of A118. The integrases from phages Bxb1, φFC1, and φRV1 carried out such recombination at their attBand attPsites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attPsite on a plasmid and pseudo attBsites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo attsites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage attsites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage attsite with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attBand attPsites at a frequency of ∼ 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attBand attPsites of A118. The integrases from phages Bxb1, φFC1, and φRV1 carried out such recombination at their attBand attPsites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attPsite on a plasmid and pseudo attBsites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo attsites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.