Cathepsin G is a 26,000-Da serine protease that is found in the azurophil granules of neutrophils and monocytes. The cathepsin G gene is expressed at high levels in U937 promonocytic cells, but is down-regulated with phorbol-induced differentiation. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic fibroblast library using cathepsin G cDNA, and obtained two lambda clones that contained the cathepsin G locus. The cathepsin G gene spans 2.7 kilobase pairs of genomic DNA and consists of 5 exons and 4 introns. The genomic organization of cathepsin G is similar to that of human neutrophil elastase, rat mast cell protease II, murine adipsin, and murine cytotoxic T-cell serine proteases, with protease catalytic residues located near the borders of exons 2, 3, and 5. Using in situ hybridization techniques, we localized cathepsin G to chromosome 14q11.2, a site that is near the alpha/delta T-cell receptor complex. Cathepsin G transcription is abolished in U937 nuclei with 2 micrograms/ml alpha-amanitin, indicating that this gene is probably transcribed by RNA polymerase II. The 5' end of the cathepsin G gene was defined by primer extension and S1 nuclease protection assays. A TATA box is found at position -29, and a CAAT box is found at -69 with respect to the transcription initiation site. Having defined the genomic structure and chromosomal location of cathepsin G, we are now attempting to identify the DNA elements in or near this gene that mediate its tissue and development-specific pattern of expression.