Oil sands process-affected water (OSPW) - a byproduct of the oil sands industry in Northern Alberta, Canada - is currently stored in on-site tailings ponds. The goal of the present study was to investigate the interaction of OSPW with the olfactory system and olfactory-mediated behaviours of fish upon the first encounter with OSPW. The response of rainbow trout (Oncorhynchus mykiss) to different concentrations (0.1, 1, and 10%) of OSPW was studied using a choice maze and electro-olfactography (EOG), respectively. The results of the present study showed that rainbow trout are capable of detecting and avoiding OSPW at a concentration as low as 0.1%. Exposure to 1% OSPW impaired (i.e. reduced sensitivity) the olfactory response of rainbow trout to alarm and food cues within 5 min or less. The results of the present study demonstrated that fish could detect and avoid minute concentrations of OSPW. However, if fish were exposed to OSPW-contaminated water and unable to escape, their olfaction would be impaired.