Nucleotide sequence analyses of human coproporphyrinogen oxidase (CPO) have shown two possible translation initiation eodons (TICs) in the region upstream from the region coding the mature polypeptide. These TICs lead to the precursor polypeptides of CPO (long and short pCPOs), which have 110 and 10 amino acids (long and short) presequences, respectively. To determine which TIC is the real one, import reactions to mitochondria of the two pCPOs synthesized using in vitro transcription-translation system were analyzed. The results showed that the long pCPO was imported into mitochondria and sorted properly to the intermembrane space, whereas the short pCPO was not. We also found several miss-readings in the reported nucleotide sequence of mouse CPO gene. This correction revealed that mouse pCPO has also a longer presequence, which are very similar to the long presequence of human. These results indicate that the long, but not short, presequence is the real one.