We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses lambda-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.