The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5'-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter-luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.