Exposure to certain environmental contaminants such as agricultural pesticides can alter normal endocrine and reproductive parameters in wild fish populations. Recent studies have found widespread pesticide contamination across the rivers that discharge into the Great Barrier Reef lagoon. Potential impacts on native fish species exposed to known endocrine disrupting chemicals such as atrazine, simazine, and diuron have not been assessed. In the present study, the authors examined the endocrine and physiological effects of short-term, acute exposure of environmentally relevant concentrations of analytical grade atrazine in juvenile barramundi (Lates calcarifer) in a controlled laboratory experiment. Expression of hepatic vitellogenin was not affected, supporting results of previous studies that showed that atrazine does not have a direct estrogenic effect via mediation of estrogen receptors. The lack of effect on brain cytochrome P19B (CYP19B) expression levels, combined with increases in testosterone (T) and 17β estradiol and a stable T:17β estradiol ratio, does not support the hypothesis that atrazine has an indirect estrogenic effect via modulation of aromatase expression. Gill ventilation rate, a measure of oxidative stress, did not change in contrast to other studies finding enhanced osmoregulatory disturbance and gill histopathology after atrazine exposure. To more closely reflect field conditions, the authors recommend that laboratory studies should focus more on examining the effects of commercial pesticide formulations that contain additional ingredients that have been found to be disruptive to endocrine function.