Two predominant splice variants of the leptin receptor (LEPR) are coexpressed in leptin-responsive tissues: the long form, LEPRb, characterized as the signal-transducing receptor, and the signaling-defective short form, LEPRa. It is unknown whether heterodimers of these isoforms are capable of signal transduction via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. To address this question, chimeric receptors were constructed consisting of the transmembrane and intracellular parts of LEPRb and LEPRa fused with the extracellular domains of either the alpha- or beta-subunit of the IL-5 receptor. This strategy allows the directed heterodimerization of different LEPR cytoplasmic tails and excludes homodimerization. In COS-7 and HEPG2 cells, chimeric receptor heterodimers of LEPRa and LEPRb failed to activate the JAK/STAT pathway, whereas receptor dimers of LEPRb gave rise to the expected ligand-dependent activation of JAK2, phosphorylation of STAT3, and STAT3-dependent promoter activity. Markedly lower amounts of JAK2 were found to be associated with immunoprecipitated LEPRa chimeras than with LEPRb chimeras. Analysis of a series of deletion constructs indicated that a segment of 15 amino acids in addition to the 29 amino acids common to LEPRa and LEPRb was required for partial restoration of JAK/STAT activation. Site-directed mutagenesis of the critical sequence indicated that two hydrophobic residues (Leu896, Phe897) not present in LEPRa were indispensable for receptor signaling. These findings show that LEPRa/LEPRb heterodimers cannot activate STAT3 and identify sequence elements within the LEPR that are critical for the activation of JAK2 and STAT3.