Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes. Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption. Gene replacement requires homologous recombination at the target locus. The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette. Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism. PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae. Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges. The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species. The high capacity of in vivo recombination of Sac. cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations. This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions. Furthermore, laborious cloning procedures, e.g. the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit.