Background: Exposure to traffic-related air pollution (TRAP) is associated with accelerated cognitive aging and higher dementia risk in human populations. Rodent brains respond to TRAP with activation of astrocytes and microglia, increased inflammatory cytokines, and neurite atrophy. A role for Toll-like receptor 4 (TLR4) was suggested in mouse TLR4-knockouts, which had attenuated lung macrophage responses to air pollution. Methods: To further analyze these mechanisms, we examined mixed glial cultures (astrocytes and microglia) for RNA responses to nanoscale particulate matter (nPM; diameter <0.2 mu m), a well-characterized nanoscale particulate matter subfraction of TRAP collected from a local freeway (Morgan et al. Environ Health Perspect 2011; 119,1003-1009, 2011). The nPM was compared with responses to the endotoxin lipopolysaccharide (LPS), a classic TLR4 ligand, using Affymetrix whole genome microarray in rats. Expression patterns were analyzed by significance analysis of microarrays (SAM) for fold change and by weighted gene co-expression network analysis (WGCNA) to identify modules of shared responses between nPM and LPS. Finally, we examined TLR4 activation in hippocampal tissue from mice chronically exposed to nPM. Results: SAM and WGCNA analyses showed strong activation of TLR4 and NF-kappa B by both nPM and LPS. TLR4 siRNA attenuated TNF alpha and other inflammatory responses to nPM in vitro, via the MyD88-dependent pathway. In vivo, mice chronically exposed to nPM showed increased TLR4, MyD88, TNF alpha, and TNFR2 RNA, and decreased NF-kappa B and TRAF6 RNA TLR4 and NF-kappa B responses in the hippocampus. Conclusions: These results show TLR4 activation is integral in brain inflammatory responses to air pollution, and warrant further study of TLR4 in accelerated cognitive aging by air pollution.