This study was conducted to evaluate whether a laboratory exposure of reference fish to oil sands sediment could produce biochemical responses (increases in 7-ethoxyresorufin-O-deethylase [EROD] activity and decreases in vitro steroid production capacity) similar to fish caught in the Athabasca Oil Sands area. Sediment samples from the Steepbank River, Alberta, were collected outside of the oil sands area at a reference site (S-Ref), within the oil sands areas where oil sand compounds leach naturally into the surface water (S-Nat), and within the natural-leach deposit areas, but also adjacent to anthropogenic mining activity (S-Dev). In the laboratory, an Ontario reference population of slimy sculpin (Cottus cognatus) were exposed to sediment concentrations of 10 or 20 g/L, for 4 and 8 d. A period of 4 d was sufficient to induce EROD activity in this species by these sediments. The EROD activity measured in exposed fish was comparable to that measured in fish native to the oil sands area. This study was not capable of predicting a reduced ability of gonadal tissue of exposed fish to produce steroid hormones in vitro, as was demonstrated in the wild fish assessment of 1999 and 2000. The short-term laboratory bioassay exposing slimy sculpin to sediment from the Athabasca Oil Sands area was not a suitable surrogate for field studies, however it could be an important tool in identifying MFO-inducing compounds in Athabasca Oil Sands sediment using a Toxicity Identification Evaluation (TIE).