With the completion of genome-sequencing projects, intentional modification of definite bacterial artificial chromosomes (BACs), which carry entire components of most eukaryotic genes, is becoming more important for the subsequent functional genomics studies. The newly optimized Red/ET recombination system was applied to the BAC modification. Mediated by the plasmid, pSC101-BAD-gbaA, and assisted by the counter-selectable/selectable system conferred by rpsL-Neo, two BACs were successfully modified. One step selectable BAC modification such as simple deletion or insertion was achieved in one week. For insertion or fusion of unselectable gene such as Cre, EFGP and LacZ as well as point mutation into targeted BACs, two rounds of Red/ET recombination was proceeded and two weeks was needed. After L-arabinose induction for transient expression of redγ/redβ/redα/recA, the pSC101-BAD-gbaA plasmid died out spontaneously from the BAC host bacteria by shifting the temperature from 30℃ to 37℃ . Thus, there was no DNA contamination in the modified BACs being used for subsequent transgenesis research. The high efficient BAC modification mediated by optimized Red/ET recombination offers a significant facility to the functional genomics investigations.