We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46. These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi. In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences. In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae.