Corticosteroid hormones, including the mineralocorticoids and the glucocorticoids, regulate diverse physiological functions in vertebrates. These hormones act through two classes of corticosteroid receptors (CR) that are ligand-dependent transcription factors: type I or mineralocorticoid receptor (MR) and type II or glucocorticoid receptor (GR). There is substantial overlap in the binding of these two receptor types to hormones and to DNA. In fish, the overlap in processes controlled by CRs may be different from that in other vertebrates, as fish are thought to synthesize only glucocorticoids, whereas they express both GR and MR. Here we describe the characterization of four CRs in a cichlid fish, Haplochromis burtoni: a previously undescribed GR (HbGR1), another GR expressed in two splice isoforms (HbGR2a and HbGR2b), and an MR (HbMR). Sequence comparison and phylogenetic analysis showed that these CRs sort naturally into GR and MR groups, and that the GR duplication we describe will probably be common to all teleosts. Quantitative PCR revealed differential patterns of CR tissue expression in organs dependent on corticosteroid action. Trans-activation assays demonstrated that the CRs were selective for corticosteroid hormones and showed that the HbMR was similar to mammalian MRs in being more sensitive to both cortisol and aldosterone than the GRs. Additionally, the two HbGR2 isoforms were expressed uniquely in different tissues and were functionally distinct in their actions on classical GR-sensitive promoters. The identification of four CR subtypes in teleosts suggests a more complicated corticosteroid signaling in fish than previously recognized.