The transcriptional response of adipose tissue depots with respect to their immune responsiveness in dairy cows remains largely unknown. Thus, we examined mRNA expression and responsiveness of subcutaneous (SUB) and mesenteric (MES) adipose tissue from nonpregnant dairy cows to a short-term (2 h), in vitro lipopolysaccharide (LPS) challenge (20 microg/mL in physiological saline). Abundance of mRNA for tumor necrosis factor-alpha (TNFA), interleukin-6 (IL6), serum amyloid A3 (SAA3), toll-like receptor 4 (TLR4), monocyte chemoattractant protein-1 (CCL2), and RANTES/chemokine C-C motif ligand 5 (CCL5) were analyzed using quantitative polymerase chain reaction (PCR) from tissue samples collected at slaughter from 5 nonpregnant/nonlactating Holstein cows. Prior to LPS challenge, SAA3 mRNA abundance was greater in MES than SUB tissue. Regardless of depot site, LPS led to greater mRNA abundance of TNFA and IL6 and was more pronounced for IL6 in MES. We also observed a marked increased in expression of CCL2, CCL5, TLR4, IL6, and TNFA in both MES and SUB during the 2-h incubation with saline alone (ie, the control). Because mRNA expression of the apoptotic markers B-cell CLL/lymphoma 2 (BCL2) and tumor protein p53 (TP53) did not differ during the 2-h incubation, it is less likely that the response to saline was a result of increased rate of cell death during incubation. Analysis using semiquantitative PCR of the 16s rRNA gene in cDNA from tissue explants revealed the presence of bacteria likely arising from contamination during sample collection. Furthermore, surfactant medium from about 50% of explant cultures had viable aerobic bacteria without differences between treatments or tissue samples. Thus, the presence of bacteria could partly explain the large increase in inflammatory-related genes after 2-h incubation with saline. The higher SAA3 expression in MES suggests that this acute-phase protein has a role in lipid metabolism and/or transport during an immune challenge. Overall, results provided evidence that adipose depots of dairy cows are capable of synthesizing chemokines and are immune responsive when exposed to inflammatory conditions that can arise from a pathogenic insult or during and soon after parturition.