Leptin and the leptin receptor are key players in the regulation of body weight. In an attempt to dissect the molecular mechanism of the Zucker fatty rat leptin receptor mutation (Gln269 --> Pro) we analyzed the effects of this mutation on leptin receptor signaling and expression in three different expression systems: 1) 32D cells expressing leptin/erythropoietin receptor chimeras, 2) COS-7 cells expressing a leptin receptor short form, and 3) 293 cells expressing soluble receptor forms. To determine if the Gln269 --> Pro mutation is critical for the observed phenotype, we made a similar Gln --> Pro mutation at a vicinal residue two amino acids upstream of the fatty mutation to see if it would have similar effects. Incorporation of either of the Gln --> Pro mutations into wild type receptor forms did not interfere with leptin binding, but it resulted in a signaling-incompetent receptor. In addition, the majority of the mutant receptor protein was localized intracellularly. Our results suggest that the obese phenotype resulting from the Gln269 --> Pro mutation in the leptin receptor of the Zucker fatty rat may be due not only to a reduced cell surface expression of this form of the leptin receptor, but also to a post-leptin binding malfunction of the receptor that interferes with subsequent signal transduction.