The host response to adenovirus (Ad) infection involves induction of cytokines in both hemopoietic cells and lung epithelia. This is accompanied by an initial neutrophilic alveolitis. We have demonstrated induction of the lung neutrophil chemokine IL-8 by Ad7, a major lung pathogen, in A549 lung epithelial cells and lung tissue in an Erk (extracellular-signal-regulated-kinase)-dependent manner. We wished to determine the mechanism of chemokine induction from the standpoint of the Ad infectious cycle in the epithelial cell, and whether Erk induction is sufficient for this process. A549 cells were exposed to Ad treated with psoralen/UV irradiation, which terminates the infectious cycle prior to viral gene expression. This treatment significantly diminished viral gene expression but not IL-8 gene induction as measured by semiquantitative RT-PCR. We then inhibited internalization of Ad by treatment of cells with EGTA in the presence of calcium-free media. The treatment inhibited internalization by 81% as determined by FACS for Ad hexon in the presence or absence of trypsin, and as visually determined by confocal microscopy. We next measured the effect of inhibition of internalization by EGTA + calcium free conditions on IL-8 induction and Erk induction. PMA (100 ng/mL) was used as a positive control, virus-free buffer as a negative control. Ad7 and PMA induced IL-8 release by 3-4 fold and 12-22 fold respectively over buffer-exposed cells. Conditions preventing Ad internalization inhibited IL-8 induction 70-80% by Ad7, but only 8-37% by PMA. The same conditions transiently delayed, but did not significantly inhibit Erk induction by Ad7. The results demonstrate that inhibition of Ad internalization, but not viral gene expression, prevents the cellular response of IL-8 induction by Ad. The data also show that Erk induction occurs prior to internalization and is required for IL-8 induction. Thus the site of induction of IL-8 by Ad is internal to the cell membrane, but external to the nucleus. In addition, Erk induction, while necessary, is not sufficient for IL-8 induction by Ad.