Toll-like receptors (TLRs) play an important role in the innate immune response of avian heterophils. We previously used the pharmacological inhibitors genistein, verapamil, chelerythrine, and pertussis toxin to investigate the upstream signaling events involved in TLR2-mediated oxidative burst in chicken heterophils. Only chelerythrine, a protein kinase C inhibitor, was found to significantly inhibit oxidative burst stimulated by the TLR2 agonist lipoteichoic acid (LTA). In the present study, we used selective pharmacological inhibitors to investigate the roles of phosphatidylinositol-3'-kinase (PI3-K), phospholipase C (PLC), calcium-dependent protein kinase C (PKC), extra-cellular signal regulated kinase (ERK), and nuclear translocation factor kappa B (NF-kappaB) on TLR2-mediated oxidative burst. U-73122 (a PLC inhibitor), wortmannin (a PI3-K inhibitor), PD 98059 (an ERK inhibitor), Gö 6976 (a PKC inhibitor) and Bay 11-7082 (a NF-kappaB inhibitor) significantly decreased LTA-stimulated oxidative burst in heterophils by 77%, 30%, 36%, 78%, and 61%, respectively. Activated TLR2 utilizes PI3-K, PLC, PKC, ERK, and NF-kappaB as signaling factors that mediate the oxidative burst of chicken heterophils.