ICAM-1 expression contributes to lung inflammation in animals exposed to hyperoxia and endotoxin, and the details of ICAM-1 regulation are not elucidated. Previously, we cloned and sequenced 2.5 kb of the 5' flanking sequence of murine ICAM-1 and carried out a computer search for known cis elements and found a concentration of elements within 400 bp of the start site. However, transient transfection studies and gel mobility shift assays did not reveal signficant regulation of the gene within this 400 bp sequence. The purpose of this study was to determine whether there was regulation of the murine ICAM-1 promoter from -400 to -2500 in the 5' flanking sequence. We created serial deletion constructs ligated to the luciferase gene, and transfected cells with these deletion constructs. After transfections, we exposed cells to TNF-α and measured luciferase activity after the exposures. We found that the region of the 5' flanking sequence from -2454 to-1462 had more than double the luciferase activities in transfected cells exposed to TNF-α, whereas the -1406 promoter construct did not induce luciferase activity in transfected cells exposed to TNF-α. In conclusion, the TNF-α inducibility of the -1462-luciferase construct with the lack of inducibility in the -1406-luciferase construct suggests that the DNA sequence between these two constructs contains elements crucial for the regulation of ICAM-1 gene, although the precise nature the regulation is not known at this time.