The generation of unmarked deletion mutants free from polar effects on downstream genes is typically a lengthy and arduous process in Streptomyces spp. The use of FLP recombinase can greatly facilitate this process when combined with established polymerase chain reaction (PCR)-targeting techniques. In vivo production of FLP within Streptomyces cells would streamline the process further, but expression of flp in Streptomyces spp. has proven difficult to achieve. Two Escherichia coli-Streptomyces shuttle plasmids that constitutively express native flp within Streptomyces cells were constructed and tested within Streptomyces clavuligerus and Streptomyces coelicolor to produce in-frame mutations in genes associated with antibiotic production. Only one of the flp-expressing plasmids was functional in S. clavuligerus, but both functioned in S. coelicolor and both were easily lost from cells. Although a separate study has recently shown successful expression of a synthetic flp gene in Streptomyces, this is the first report of expression of the native flp gene within Streptomyces spp. Through the use of these plasmids to generate unmarked deletion mutants, C7p was shown to be essential for production of 5S clavams in S. clavuligerus, and RedJ was demonstrated to be important for optimal undecylprodigiosin biosynthesis in S. coelicolor but traces of the antibiotic were still produced in a DeltaredJ mutant.