We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.