ICAM-1 expression has been found to be important in the development of lung inflammation and injury. We have reported increased ICAM-1 expression in lungs of mice exposed to hyperoxia and lipopolysacharide (LPS). To further explore the mechanisms of ICAM-1 upregulation in LPS exposed lungs, we have sequenced approximately 2.5 kb of the 5' flanking sequence of murine ICAM-1 and have found that the region between -398 and -280 from the start site contain a number of cis-elements known to regulate gene expression. We examined this region of the 5' flanking sequence of ICAM-1 by transfecting cells with serial deletions of the sequence ligated to the reporter gene, chloramphenicol acetyl transferase (CAT) (-398CAT, -355CAT, -309CAT and -280CAT). The cells used were A549 cells, which are transformed cells with characteristics of Type II cells, and in which we found increased endogenous ICAM-1 mRNA when exposed to LPS. After transfections, we exposed cells to 0, 10, or 50 μg/ml of LPS for 8 h, after which we measured CAT expression in cell lysates. We found that in cells exposed to vehicle alone that CAT expression nearly doubled from -398CAT to-355CAT (P<0.05). In addition, when cells transfected with -355CAT were exposed to LPS, CAT expression was approximately doubled when compared to cells exposed to vehicle only (P<0.05). LPS inducibility observed in the - 355CAT construct was not observed in any of the other deletional constructs. One possible explanation for the LPS inducibility of the -355CAT construct is that LPS induces TNF-α and/or IL-1ß in LPS-exposed cells, which then acts on the promoter to increase CAT activity. To test this hypothesis we measured these cytokines in cells exposed to LPS for 1, 2 or 4 h, and found no measurable cytokines in the cell supernatants. In conclusion, our findings suggest that there is a DNA-protein interaction(s) in the segment of DNA between -398 and -355 that down regulates basal ICAM-1 expression, and the LPS inducibility observed in cells transfected with -355CAT, but not in -309CAT suggests that there is a protein-DNA interaction(s) between -355 and -309 that confers an increase in ICAM-1 expression in response to endotoxin exposure. Supported by NIH grant 5 P30 HD27823-04.