The goal of this study was to determine the relationship between cortisol and the toadfish serotonin 2A (5-HT2A) receptor, which is believed to be responsible for the activation of the toadfish urea transporter, tUT. We hypothesize that elevations in cortisol would play a role in the regulation of the 5-HT2A receptor at the level of mRNA expression, ligand binding, and/or function. To test this idea, cortisol levels were manipulated by either crowding or through treatment with the cortisol synthesis blocker, metyrapone. Crowded fish had significantly higher circulating cortisol levels compared to uncrowded fish and cortisol levels in metyrapone-treated fish were significantly lower than saline-treated controls. No significant difference was measured in gill 5-HT2A mRNA expression levels between uncrowded and crowded, control- or metyrapone-treated fish. Furthermore, no significant difference was measured in [3H]-5-HT binding kinetics or in the competitive binding of the 5-HT2 agonist, α-methyl 5-HT, to isolated gill basolateral membranes of uncrowded or crowded toadfish. However, the binding maximum (Bmax) of the 5-HT2A receptor antagonist, [3H]-ketanserin, was significantly different between all four groups of fish (metyrapone > control > crowded > uncrowded). Furthermore, metyrapone-treated fish excreted approximately twofold more urea compared to controls when injected with α-methyl 5-HT, a 5-HT2 receptor agonist shown to stimulate urea excretion. Our results suggest that cortisol may have differential effects on 5-HT receptor binding, which could have potential implications on the control of pulsatile urea excretion in toadfish. J. Exp. Zool. 319A:237–248, 2013. © 2013 © 2013 Wiley Periodicals, Inc.