The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.