Cortisol is a glucocorticoid hormone which is an endocrine signaling molecule in all vertebrates and acts through intracellular glucocorticoid receptors (GRs). Cortisol affects many biological functions including immunity, stress, growth, and reproduction. The objective of this study was to investigate the ontogeny of the cortisol and GR stress response in channel catfish (Ictalurus punctatus) at several early life stages. To accomplish this, resting and stress-induced levels of tissue cortisol and the two catfish GRs (GR-1 and GR-2) expression were measured. Resting cortisol levels in newly fertilized eggs averaged 2.4 ± 0.2 ng/egg and decreased to 0.4 ± 0.01 ng/egg by day 5. Cortisol levels in newly fertilized eggs subjected to an acute stress (lowered dissolved oxygen from 6.5 mg/L to 1.8 mg/L) averaged 2.3 ± 0.1 ng/egg and decreased to 0.3 ± 0.03 ng/egg by day 5. At hatching, resting cortisol levels were 24 ± 1.0 ng/0.1 g tissue while levels increased to 83 ± 2.0 ng/0.1 g tissue in fry subjected to an acute stress (P < .05). Four days post-hatch, resting cortisol levels were 83 ± 1.0 ng/0.1 g tissue while levels increased to 149 ± 4.0 ng/0.1 g tissue in fry subjected to an acute stress (P < .01). There was no significant difference between GR-1 and GR-2 mRNA in stressed and unstressed newly hatched fry. Four days post-hatch, GR-1 mRNA increased 3-fold while GR-2 mRNA increased 2-fold in fry that were subjected to low dissolved oxygen conditions (P < .05). These results indicate that cortisol biosynthesis, integration and maturation of the hypothalamic-pituitary-interrenal (HPI) axis can be observed in channel catfish at hatching. The upregulation of GR-1 and -2 mRNA in stressed fry supports roles for both transcripts in integrating the channel catfish stress response.