Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants, because there are only two efficient resistance markers available for allelic exchange experiments. We have established a system based on the Flp recombinase of the yeast Saccharomyces cerevisiae for use in the nonpathogenic model organism Mycobacterium smegmatis. This system consists of a hygromycin resistance cassette flanked by two Flp recognition targets (FRT) in direct orientation and a curable plasmid for expression of the flp gene. The FRT-hyg-FRT cassette was used on a suicide plasmid and on a conditionally replicating plasmid to delete two of the four known porin genes of M. smegmatis, mspA and mspC, respectively, by homologous recombination. The hyg gene was specifically removed from the chromosome of both mutants upon expression of the flp gene. Based on the marker-less mspC mutant strain, a double knock-out mutant lacking also mspA was obtained using the same strategy. Thus, by a fast and efficient two-step procedure, each of the porin genes was replaced by a single FRT site, which can be further used for site-specific integration. These results show that the Flp/FRT system is a suitable genetic tool for constructing unmarked mutations and for the analysis of redundant genes by consecutive gene deletions in M. smegmatis. © 2004 Elsevier B.V. All rights reserved.