Exposure to air pollutants such as ozone (O₃) induces airway hyperresponsiveness (AHR) and airway inflammation. Toll-like Receptors (TLR) are first-line effector molecules in innate immunity to infections and signal via adapter proteins, including myeloid differentiation factor-88 (MyD88). We investigated the sensing of ozone by TLR2, TLR4 and MyD88. Ozone induced AHR in wild-type (WT) C57BL/6 mice, but AHR was absent in TLR2^-/-, TLR4^-/- and MyD88^-/- mice. Bronchoalveolar lavage (BAL) neutrophilia induced by ozone was inhibited at 3 hr but not at 24 hr in TLR2 and TLR4^-/- mice, while in MyD88^-/- mice, this was inhibited at 24 hr. We investigated the expression of inflammatory cytokines, and TLR2, TLR4 and MyD-88 in these mice. Ozone induced time-dependent increases in inflammatory gene expression of KC and IL-6, and of TLR2, TLR4 and MyD88 in WT mice. IL-6 and KC expression induced by ozone was inhibited in TLR2^-/-, TLR4^-/- and MyD88^-/- mice. Expression of MyD88 was increased in TLR2 and TLR4^-/- mice, whilst induction of TLR2 or TLR4 was reduced in TLR2^-/- and TLR4^-/- mice, respectively. TLR2 and TLR4 mediate AHR induced by oxidative stress such as ozone, while the adapter protein MyD88, but not TLR2 or TLR4, is important in mediating ozone-induced neutrophilia. TLR2 and TLR4 may also be important in regulating the speed of neutrophilic response. Therefore, ozone may induce AHR and neutrophilic inflammation through the activation of the toll-like receptor pathway that may sense non-infectious stimuli such as oxidative stress.