Background: Ambient particulate matter (PM) smaller than 2.5 µm in diameter (PM_2.5) undergoes diurnal changes in chemical composition due to photochemical oxidation. In this study we examine the relationships between oxidative activity and inflammatory responses associated with these diurnal chemical changes. Because secondary PM contains a higher fraction of oxidized PM species, we hypothesized that PM_2.5 collected during afternoon hours would induce a greater inflammatory response than primary, morning PM_2.5. Methods: Time-integrated aqueous slurry samples of ambient PM_2.5 were collected using a direct aerosol-into-liquid collection system during defined morning and afternoon time periods. PM_2.5 samples were collected for 5 weeks in the late summer (August-September) of 2016 at a central Los Angeles site. Morning samples, largely consisting of fresh primary traffic emissions (primary PM), were collected from 6-9am (am-PM_2.5), and afternoon samples were collected from 12-4pm (pm-PM_2.5), when PM composition is dominated by products of photochemical oxidation (secondary PM). The two diurnally phased PM_2.5 slurries (am- and pm-PM_2.5) were characterized for chemical composition and BV-2 microglia were assayed in vitro for oxidative and inflammatory gene responses. Results: Contrary to expectations, the am-PM_2.5 slurry had more proinflammatory activity than the pm-PM_2.5 slurry as revealed by nitric oxide (NO) induction, as well as the upregulation of proinflammatory cytokines IL-1β, IL-6, and CCL2 (MCP-1), as assessed by messenger RNA production. Conclusions: The diurnal differences observed in this study may be in part attributed to the greater content of transition metals and water-insoluble organic carbon (WIOC) of am-PM_2.5 (primary PM) vs. pm-PM_2.5 (secondary PM), as these two classes of compounds can increase PM_2.5 toxicity.