Two types of vacuolar sorting signals (VSSs), an asparagine-proline-isoleucine-arginine-leucine (NPIRL)-related VSS in the N-terminal propeptides (NTPPs) and a C-terminal VSS in the C-terminal propeptides (CTPPs), function differently in plant cells. A precursor to a 20-kDa protein of potato tuber (PT20) contains two NPIRL-related sequences, NPINL in a short NTPP and NPLDV close to the C terminus of the precursor. We made mutant forms of sweet potato sporamin (SPO), nPT20-SPO, in which the N-terminal pre-pro part was exchanged with that of the precursor to PT20, and SPO-PT20c, in which the C-terminal 13 amino acids of the precursor to PT20 was attached to the C terminus of delta pro-SPO which lacked NTPP. Both nPT20-SPO and SPO-PT20c were efficiently transported to the vacuoles in tobacco cells. Unlike nPT20-SPO, the vacuolar transport of SPO-PT20c was inhibited by wortmannin and by the C-terminal addition of Gly or Gly-Gly suggesting its similarity to the vacuolar transport of sporamin mediated by CTPP of barley lectin. Further analysis of the C-terminal sequence of PT20 indicated that the most C-terminal SFKQVQ sequence functions as the C-terminal VSS. These results suggest that the precursor to PT20 contains both NPIRL-like VSS in its NTPP and C-terminal VSS at the C terminus.