The corticotropin-releasing factor (CRF) system in fish functions to maintain homeostasis during stress in part by regulating cortisol production via the hypothalamus-pituitary-interrenal (HPI) axis. Towards understanding the role of the CRF system in vertebrate development, we describe the ontogeny of the CRF system, cortisol, and the stress response in the zebrafish, Danio rerio. Early embryonic expression of mRNA encoding CRF, urotensin I (UI), CRF-binding protein (CRF-BP), and two CRF receptors (CRF-R1 and CRF-R2) suggest a function in the early organization of the developing embryo. The expression patterns of CRF, UI, and CRF-BP in the larval brain are consistent with the adult distribution patterns for these genes and support HPI-axis independent functions. The relative amounts of CRF and UI mRNA in the heads and tails of developing and adult zebrafish suggest that CRF functions primarily in the brain while UI also plays an important role in the caudal neurosecretory system. The amount of cortisol in developing zebrafish is low and relatively constant through the first 6 days of development. The commencement of feeding after 4 dpf, however, significantly increases basal cortisol production. Finally, we show that zebrafish larvae are able to respond to an osmotic stressor as early as 3 dpf. Overall, results from this study establish the zebrafish as a model species for research on stress during ontogeny and offer new insights into an HPI-axis independent function for the CRF system during embryogenesis.