In mammals, leptin (Lep) binding proteins (LepBPs) derived from Lep receptor (LepR) gene or protein bind most of the circulating Lep, but to date, information on LepBPs in nonmammalian vertebrate classes is lacking. This study details the characterization of multiple LepBPs in rainbow trout (Oncorhynchus mykiss), an early poikilothermic vertebrate, and presents the complete coding sequences for 3 of them. Size-exclusion chromatography and cross-linking assay identified plasma proteins bound to Lep ranging from 70 to 100 kDa. LepBPs were isolated from plasma by affinity chromatography, and their binding specificity was assessed by a competitive binding assay. A RIA for LepBPs indicates that plasma LepBP levels decline after fasting for 3 weeks. Immunoblotting of LepBPs using antibodies against different LepR epitopes shows that the LepBPs are indeed LepR isoforms. The alternatively spliced LepR transcripts (LepR(S1-3)) that include only the extracellular segment transcribe the 90-kDa LepBP1, the 80-kDa LepBP2, and the 70-kDa LepBP3, respectively. LepR(S1) generally has lower expression than the long-form LepR in most tissues. LepR(S2) is primarily expressed in adipose tissue, whereas LepR(S3) is expressed abundantly in brain and spleen, and moderately in liver and gills. The mRNA levels of hepatic LepR(S3) increase after 2 weeks of fasting. This study demonstrates a mechanism in fish for the generation of LepBPs that differs from that seen in mammals and indicates that the physiologic action of Lep in these poikilothermic vertebrates can be modulated, both centrally and peripherally, by the differentiated, tissue-specific expression of multiple LepBPs.