In humans, two glucocorticoid receptor (GR) splice variants exist: GR and GR, which are identical between amino acids 1–727 and then diverge. Whereas GR (the canonical GR) acts as a ligand-activated transcription factor, GR does not bind traditional glucocorticoid agonists, lacks GR's transactivational activity, and acts as a dominant-negative inhibitor of GR. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR -isoform has only been detected in humans and therefore an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GR from human GR. The zebrafish GR -isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GR/GR ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GR is similar to its human equivalent. Finally, expression levels of GR and GR were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body and GR mRNA levels were relatively low compared to GR mRNA levels, as in humans. Thus, for the first time a GR -isoform has been identified in a non-human animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.