Background: Primary (POA) and secondary (SOA) organic aerosols, deriving from both anthropogenic and biogenic sources, represent a major fraction of ambient particulate matter (PM) and play an important role in the etiology of respiratory and cardiovascular diseases, largely through systemic inflammation and cellular oxidative stress. The relative contributions of these species to the inhalation burden, however, are rather poorly characterized. In this study, we measured the in vitro oxidative stress response of alveolar macrophages exposed to primary and secondary PM derived from both anthropogenic and biogenic sources. Methods: POA and SOA were generated within an oxidation flow reactor (OFR) fed by pure, aerosolized α-pinene or gasoline engine exhaust, as representative emissions of biogenic and anthropogenic sources, respectively. The OFR utilized an ultraviolet (UV) lamp to achieve an equivalent atmospheric aging process of several days. Results: Anthropogenic SOA produced the greatest oxidative response (1900 ± 255 µg-Zymosan/mg-PM), followed by biogenic (α-pinene) SOA (1321 ± 542 µg-Zymosan/mg-PM), while anthropogenic POA produced the smallest response (51.4 ± 64.3 µg-Zymosan/mg-PM). Conclusions: These findings emphasize the importance of monitoring and controlling anthropogenic emissions in the urban atmosphere, while also taking into consideration spatial and seasonal differences in SOA composition. Local concentrations of biogenic and anthropogenic species contributing to the oxidative potential of ambient PM may vary widely, depending on the given region and time of year, due to factors such as surrounding vegetation, proximity to urban areas, and hours of daylight.