ABSTRACT Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT- lacZ - aph - P lac -FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans . Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of yacF , yacG , yacH , yacK ( cueO ), yacL , ruvA , ruvB , yabB , and yabC made with the cassette were used to verify its properties.