Infection of the bovine mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Greater understanding of the initial host response to infection may lead to more accurate selection of resistant animals or to novel prophylactic or therapeutic intervention strategies. The epithelial cell plays a role in the host response by alerting the immune system to the infection and providing a signal as to where the infection is located. To understand this process better, a cDNA microarray approach was used to search for potential signals produced by mammary epithelial cells in response to exposure to Escherichia coli lipopolysaccharide (LPS). Total RNA from separate cultures of epithelial cells from 4 Holstein cows was harvested 6 h after LPS challenge or control conditions. For each cow, RNA from control or LPS-exposed cells was transcribed to cDNA and labeled with Cy3 or Cy5, then pooled and applied to a bovine total leukocyte (BOTL) microarray slide containing 1278 unique transcripts. Dye reversal was used so that RNA from two of the control cultures was labeled with Cy3 while RNA from the other two control cultures was labeled with Cy5. From the resulting microarray data we selected 4 of the 9 genes significantly (P < 0.02) induced (>1.25-fold) in response to LPS exposure for more detailed analysis. The array signal intensity for 3 of these genes, RANTES/CCL5, IL-6 and T-PA, was relatively low, but quantitative real-time RT-PCR (Q-RT-PCR) analysis revealed that they were induced 208-fold, 10-fold and 3-fold, respectively. The gene that showed the greatest fold induction by microarray analysis (2.5-fold) was CXCL5. This gene had a relatively strong signal intensity on the array and was easily detected by northern blot analysis, which indicated a 10-fold induction. This cell culture model system provides evidence for an important role of the mammary epithelial cell in initiating the innate response to infection.