<b>Background: </b>5 Lipoxygenase (5–LO) is a critical enzyme in the production of the leukotrienes. We have identified a series of mutations in the 5–LO gene that modify gene transcription. These mutations consist of addition of an Sp–1 binding motif (–GGGCGG–) or deletion of one or two Sp–1 binding motifs in the 5–LO core promoter. <b>Methods: </b>Mutant forms of the 5–LO core promoter were placed in a chloramphenicol acetyl transferase (CAT) reporter construct using either HeLa or SL–2 cells. <b>Results: </b>In HeLa cells all of the mutant forms are less effective in driving CAT reporter activity than the wild–type promoter. In SL–2 cells the construct containing the addition mutation was more effective in driving CAT reporter activity, while the constructs containing the deletion mutations were less effective. <b>Conclusions: </b>These data indicate that naturally occurring mutations in the 5–LO core promoter modify gene transcription in vitro.