<jats:title>ABSTRACT</jats:title> <jats:p> The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera <jats:named-content content-type="genus-species">Streptomyces</jats:named-content> , <jats:named-content content-type="genus-species">Micromonospora</jats:named-content> , and <jats:named-content content-type="genus-species">Saccharothrix</jats:named-content> . Two different systems based on Cre/ <jats:italic>loxP</jats:italic> and Dre/ <jats:italic>rox</jats:italic> have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific <jats:italic>loxLE</jats:italic> and <jats:italic>loxRE</jats:italic> sites was similar to its activity on wild-type <jats:italic>loxP</jats:italic> sites. Moreover, an apramycin resistance marker flanked by the <jats:italic>loxLERE</jats:italic> sites was eliminated from the <jats:named-content content-type="genus-species">Streptomyces coelicolor</jats:named-content> M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from <jats:named-content content-type="genus-species">Streptomyces</jats:named-content> sp. strain Tü6071, <jats:named-content content-type="genus-species">Streptomyces cinnamonensis</jats:named-content> A519, and <jats:named-content content-type="genus-species">Streptomyces aureofaciens</jats:named-content> Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.