Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.