The main objective of this study was to evaluate the influence of naturally occurring oil sands-related compounds (OSRC) on reproductive function in fish in order to assess the impacts of anthropogenic point-source inputs. The health of slimy sculpin (Cottus cognatus) and pearl dace (Semotilus margarita) collected from the Alberta Athabasca Oil Sands (Canada) watershed were examined. Two rivers were selected for study: the Steepbank and the Ells. These rivers originate outside the oil sands formation, where fish are unexposed (Ref), exposed to naturally occurring oil sands-related compounds (Nat), or exposed to naturally occurring compounds as well as adjacent to surface mining activity (Dev). Assessment endpoints included gonadosomatic indices (GSI), fecundity, and in vitro gonadal steroid production. In vitro gonadal incubations demonstrated lower levels of steroid production at sites along the Steepbank River within the oil sands deposit. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity, an indicator of exposure to OSRC, was elevated twofold at the site with natural compounds and up to 10-fold at the site adjacent to development compared to EROD activity in fish from the reference site. Fish collected in the Ells River had a threefold induction in EROD activity but no significant reduction in steroid production when compared to reference fish. No consistent alterations in gonadal development were seen in fish collected from sites within the oil sands deposit. This research in the Athabasca River basin provides baseline information of the health of fish populations within the oil sands deposit prior to further development in the area.