Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.