The goal of this study was to examine the mechanisms of leukotriene C₄ (LTC₄) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC₄ 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 LTC₄ synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC₄ synthase gene is transcriptionally regulated in mononuclear phagocytes.