The involvement of Toll-like receptors (TLRs) and other immune signalling genes during challenge of bovine macrophages with bacterial products derived from disease-causing bacteria in cattle was investigated. An in vitro cell culture model of bovine monocyte derived macrophages (MDM) was established and these cells were exposed to purified protein derivative (PPD-b) derived from Mycobacterium bovis and to lipopolysaccharide (LPS) derived from Escherichia coli. Following 24h incubation, total RNA was extracted and expression of immune related genes was determined by real time quantitative reverse transcription PCR (qRT-PCR). Expression of a selection of genes spanning the TLR-2 and TLR-4 pathways, from the initial activation of the receptors to the production of pro-inflammatory cytokines and chemokines was determined. Results from repeat experiments using MDM from seven different age-matched dairy cattle showed that PPD-b treatment caused significant up-regulation of the TLR2 and TLR4 genes and the expression profile of TLR adaptor molecules suggested that this signalling is MYD88-dependent. Conversely, LPS caused significant up-regulation of TLR4 via a MYD88-independent signalling pathway. Significant up-regulation of genes involved with NF-κB signalling was also detected in PPD-b- and LPS-treated samples accompanied by the expression of pro-inflammatory cytokine (TNF, IL1B, IL6) and chemokine genes (IL8, CCL5, CCL3). Overall, LPS challenge resulted in a more marked up-regulation of immune-related genes. Furthermore, the magnitude fold-change difference in gene expression suggests, at least in part, that bovine macrophages produce IFN-γ as a result of LPS challenge.