For many gram-negative bacteria, whose transformation systems have yet developed, following a two stage manipulation for gene cloning is a common choice. Following this strategy, DNAs are cloned in Escherichia coli, using a mobilizable vector, and the recombinant plasmids conjugally transferred into the original host. In this study, transfer of the broad-host range plasmid pLAFR1 (a 21.6 kb cosmid, TcR, derived from RK2 replicon) from E. coli to Xanthomonas campestris pv. campestris, by the help of plasmid pRK2013, was carried out to optimize the working conditions for gene cloning experiments in this phytopathogenic bacterium. Among several mating procedures tested, the highest frequencies of transfer were found by dropping the mixtures of the donor, helper and recipient cells (at ratios 1:1:10) to a nitrocellulose filter on an agar plate, with all the cells used from the cultures between OD550 0.3 to 0.5. When the non-mucoid mutant P20 was used as the recipient instead of its parental mucoid strain XC11A, 14-fold more transconjugants were obtained. In addition, the plasmid was found to be quite stable in the X. campestris cells. Further experiments showed that pLTA1, which is pLAFR1 with a cloned DNA fragment (4.4 kb) encoding alpha-amylase activity from XC11A, was not only maintained stably but also found to contribute a 8.3-fold over-production of enzyme activity to the transconjugant cells. From these studies, it has been demonstrated that cosmid pLAFR1 and the non-mucoid mutant P20 together provide a suitable vector/host system for cloning genes in X. campestris.