A carboxypeptidase E knockout (CPE KO) mouse was generated by deletion of exons 4 and 5 from the CPE gene and its phenotype was characterized. KO mice became obese by 10-12 wks of age and reached 60-80 g by 40 wks. At this age body fat content was more than double that in the wild-type (WT) controls. The null animals consumed more food overall, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than WT littermates. Fasting levels of glucose and insulin-like immunoractivity (IR) in plasma were elevated in both male and female KO mice at 20 wks; males recovered fully and females partially from this state by 32 wks. At this time, insulin-like IR in the plasma, identified as proinsulin, was 50-100 times higher than that of the WT animals. The KO mice showed impaired glucose clearance and were insulin resistant. High levels of leptin and no circulating fully processed CART, a peptide that is responsive to leptin-induced feedback inhibition of feeding, were found in serum. The KO mice were subfertile and showed deficits in GnRH processing in hypothalamus. Behavioral analyses revealed that KO animals showed diminished reactivity to stimuli, and had reduced muscle strength, coordination and visual placing, and toe-pinch reflexes. These data demonstrate that CPE KO mice display a wide-range of neural and endocrine abnormalities and suggest that CPE may have additional physiological roles beyond those ascribed to peptide processing and sorting of prohormones in cells.