Rainbow trout were exposed in situ to oil sands-affected waters for 21 d, either with or without an immune stimulation using inactivated Aeromonas salmonicida. Three aquatic systems were utilized for the experiment: a pond containing oil sands tailings capped with approximately 3 m of natural surface water, a second pond where unextracted oil sands materials were deposited in the watershed, and a reservoir receiving Athabasca River water as a reference caging location. The three systems showed a gradient of oil sands-related compounds, most notably, total naphthenic acids were highest in the system containing tailings (13 mg/L), followed by the system influenced by unextracted oil sands (4 mg/L), followed by the reference cage location (1 mg/L). Biochemical and chemical measures of exposure in rainbow trout showed the same trend, with the tailings-influenced system having the highest hepatic EROD activity and elevated bile fluorescence measured at phenanthrene wavelengths. Trout caged in the tailings-influenced location had significantly fewer leukocytes and smaller spleens as compared to the reference fish, though liver size and condition factor were unaffected. Fish in the tailings-influenced waters also demonstrated increased fin erosion, indicative of opportunistic infec- tion. The trout exposed to tailing-influenced waters also showed a significantly decreased ability to produce antibodies to the inactivated A. salmonicida. Given the complexity of the exposure conditions, exact causative agents could not be determined, however, naphthenic acids, polycyclic aromatic hydrocarbons and pH correlate with the immunotoxic effects while elevated salinity or metals seem unlikely causes.