Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element.