Ornithine decarboxylase-antizyme (Az), a polyamine-induced protein that targets ornithine decarboxylase (ODC) to rapid degradation, is synthesized as two isoforms. Studies performed in vitro indicated that the 29 and 24.5 kDa isoforms originate from translation initiation at two alternative initiation codons. Using transient transfections we demonstrate here that also in cells the two isoforms are synthesized from two AUG codons with the second being utilized more efficiently. The more efficient utilization of the second AUG is due to its location within a better sequence context for translation initiation. By using immunostaining we demonstrate that only the less expressed long form of Az is localized in the mitochondria. Moreover, this long isoform of Az and not the more efficiently expressed short isoform is imported into mitochondria in an in vitro uptake assay. Our data therefore demonstrate that a single Az transcript gives rise to two Az proteins with different N-terminal sequence and that the longer Az form containing a potential N-terminal mitochondrial localization signal is transported to mitochondria.