Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family of enzymes are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway of the cell where they are involved in the maturation of bioactive peptides. In order to determine the function of CPO, the subcellular localization of CPO was determined by immunofluorescence analysis. CPO, stably expressed in Madin-Darby canine kidney cells, was found in a punctate pattern partially associated with lipid droplets. When cells were serum-starved the structures associated with CPO became more numerous and often clustered. Addition of oleic acid to the medium caused CPO to be redistributed in a diffuse pattern with some concentration on the nuclear envelope. Western blot analysis of CPO following oleate incubation indicated an increase in the amount of a CPO isoform with lower mobility as seen by SDS-PAGE, suggestive of a post-translational modification. These changes in CPO size and distribution, dependent on nutrient availability, suggest a possible role for CPO in autophagy-related processes.