A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.