This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8-isoprostane (8-IsoP) and 8-hydroxy-2-deoxy Guanosine (8-OH-dG)], in in-vitro spent media of follicle cells. Follicles (100–130 µm) isolated from ovaries of F1 hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml–1 (control) to 45 ng ml–1] or CSC [0 µg ml–1 (control) to 130 µg ml–1]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme-linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose-dependent increase in the concentrations of 8-IsoP and 8-OH-dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke. © Her Majesty the Queen in Right of Canada 2013.