There is evidence that the combination of polycyclic aromatic hydrocarbons (PAHs) released in the Deepwater Horizon oil spill impairs the glucocorticoid stress response of vertebrates in the Gulf of Mexico, but the mechanisms are unclear. We hypothesized that inhibition of cortisol release may be due to 1) overstimulation of the hypothalamic–pituitary–inter‐renal (HPI) axis, or 2) an inhibition of cortisol biosynthesis through PAH activation of the aryl hydrocarbon receptor (AhR). Using a flow‐through system, Gulf toadfish (Opsanus beta) were continuously exposed to control conditions or one of 3 environmentally relevant concentrations of PAHs from Deepwater Horizon oil (∑PAH50 = 0–3 μg L–1) for up to 7 d. One group of toadfish was then exposed to a recovery period for up to 7 d. No changes in corticotrophin‐releasing factor mRNA expression, adrenocorticotropic hormone (ACTH), or pituitary mass suggested that overstimulation of the HPI axis was not a factor. The AhR activation was measured by an elevation of cytochrome P4501A1 (CYP1A) mRNA expression within the HPI axis in fish exposed to high PAH concentrations; however, CYP1A was no longer induced after 3 d of recovery in any of the tissues. At 7 d of recovery, there was an impairment of cortisol release in response to an additional simulated predator chase that does not appear to be due to changes in the mRNA expression of the kidney steroidogenic pathway proteins steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and 11β‐hydroxylase. Future analyses are needed to determine whether the stress response impairment is due to cholesterol availability and/or down‐regulation of the melanocortin 2 receptor. Environ Toxicol Chem 2021;40:1062–1074. © 2020 SETAC [ABSTRACT FROM AUTHOR]