Given the lack of functional transposons for use in Campylobacter spp., an alternative method of insertional mutagenesis using natural transformation was developed. High efficiencies of transformation were only obtained with species-specific DNA. This feature was a key element in the construction of mutant libraries of this bacterium. A chromosomal library of Campylobacter jejuni 81116 DNA was made in shuttle vector pUOA18. Next, a kanamycin-resistance (KmR) cassette was ligated into the inserts of the plasmids. C. jejuni 81116 was then transformed with the resulting products to allow homologous recombination between genomic fragments present in the shuttle vector and the chromosome. Transformants were pooled and chromosomal DNA from these transformants was used to retransform C. jejuni 81116. This resulted in transformants containing the KmR cassette in the chromosome but lacking the vector. In order to evaluate this approach for the construction of a mutant bank, the KmR insertional mutants were screened for loss of motility. Partial characterization of 11 non-motile mutants indicated that the inserted genes are involved in motility. Four mutants had the KmR cassette inserted in genes involved in flagella biosynthesis, namely flaA/B, neuB and flgK, and produced incomplete or no flagella. Four mutants had the KmR cassette inserted in genes possibly involved in flagella motor function: pflA, fliM and orf1 downstream of the fliN gene. Three mutants had the KmR cassette inserted in genes that are homologous to genes encoding hypothetical proteins of Helicobacter pylori.