Ornithine decarboxylase (ODC) isolated from a variety of tissues has been separated, using DEAE ion-exchange chromatography, into multiple peaks of activity that appear to be related to control of this enzyme stability. Reports of these charge isoforms in current literature are generally unclear as to whether these represent a covalent posttranslational modification or merely an alteration in structural conformation or association. In this study we investigated the relationship of this form separation to the degree of enzyme polymerization, interaction with other proteins and buffer components, and the multiple isoelectric forms of this enzyme noted in denaturing concentrations of urea. High-performance chromatography techniques were used to demonstrate that two of the major enzyme forms, ODC I and II, are really monomers of the enzyme, while minor peaks of activity frequently observed to elute after ODC II contain various dimeric enzyme states. Pyridoxal 5'-phosphate (0.05 mM) added to isolated enzyme preparations composed of I and II monomers induced the formation of I and II dimers as well as a mixed I-II dimer. All three dimer forms were observed to be natural components of freshly isolated crude cell homogenates. The charge distinction between the monomer forms I and II was found to be maintained during ion-exchange chromatography in the presence of 8 M urea, and the enzyme isoforms demonstrated distinct bands on isoelectric focusing gels run in the presence of 9 M urea. Thus, although some of the multiple ornithine decarboxylase forms identified by ion-exchange chromatography of crude mammalian cell homogenates are related to enzyme conformation, the two major forms are distinctly charged protein states that can be visualized using two-dimensional gel electrophoresis of highly purified samples.