beta-Adrenoceptors (beta-ARs) are seven-transmembrane domain, G protein-coupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported beta-ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both beta-ARs are homologs of the mammalian beta3-AR. Analysis of tissue expression patterns indicates that one of these trout beta3-adrenoceptors (beta3a-AR) is highly expressed in gill and heart, whereas the second (beta3b-AR) is highly expressed by red blood cells (RBC). Expression of the beta3b-AR in the RBC coupled with the finding of a single category of beta-AR binding sites on RBC membranes provides strong evidence for the control of the trout RBC beta-AR Na+/H+ exchanger (beta-NHE) activity by signaling through this beta3b-subtype and not through a beta1-subtype as previously proposed. The RBC-specific trout beta3b-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian beta-ARs (beta1-, beta2-, and beta3-AR). This study is the first to report the presence of a beta3-AR subtype in a fish species, and the proposal that the beta3b-AR controls RBC beta-NHE activity represents a novel role for the beta3-AR subtype in vertebrates.