The goal of this study was to examine the mechanisms of leukotriene C(4) (LTC(4)) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC(4) synthase promoter-reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between -92 and -23 bp, which contains a signal protein (Sp)1 consensus site at -42 to -37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from -56 to -17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 cell line with a construct containing the -92 to -23 bp promoter region and Sp expression vectors revealed that Sp1 and Sp3 transactivate gene transcription. We conclude that the Sp1 site is a necessary element for LTC(4) synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC(4) synthase gene is transcriptionally regulated in mononuclear phagocytes.