The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.