Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA − BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.