Phage-derived ?C31 integrase represents an attractive tool for site-directed recombination in mammalian cells. Integration is based on recombination between attachment site attB and wt/pseudo-attP'' site. Disadvantages are inefficient nonviral gene delivery and aberrant events (15% chromosomal rearrangements/deletions) within the host genome. The study''s aim was to increase safety and efficiency of ?C31 integrase. DNA binding domain was mutated by alanine scan, 22 mutants were evaluated for improved integration and intramolecular recombination. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies from 1.7 to 5.5-fold. Several mutants showed cell line-dependent integration activities. Excision assays between native attB/attP sites revealed 5 mutants with 2-fold enhanced activity. Enhanced recombination between attB and 3 described attP'' sites (hot spots) in the mammalian genome assumed preferred specificity. 2 mutants showed similar integration activity as wt due to hFIX expression in mouse hepatocytes. Mutational analysis revealed an efficient approach for improvements of integration efficiency in vitro.