Potato virus X (PVX) contains cis-acting elements including stem-loop 1 (SL1) RNA at the 5` region; SL1 is conserved among all potexviruses. The SL1 at the positive-sense RNA, SL1(+), is required for PVX RNA replication, cell-to-cell movement, and translation. Previous research demonstrated that SL1(+) RNA also serves as the origin of assembly for encapsidation of PVX RNA. To identify the essential sequences and/or regions for capsid protein (CP) subunit recognition within SL1(+) RNA, we used electrophoretic mobility shift assays (EMSA), UV cross-linking, and yeast threehybrid analyses. The EMSA and UV cross-linking analyses with PVX CP subunits and RNA transcripts corresponding to the SL1(+) RNA showed that the SL1(+) RNA formed complexes with CP subunits. We also conducted EMSA and yeast three-hybrid analyses with RNAs containing various mutations of SL1(+) RNA elements. These analyses indicated that SL1(+) RNA is required for the interaction with PVX CP and that the RNA sequences located at the loop C and tetra loop of the SL1(+) are crucial for CP binding. These results indicate that, in addition to being important for RNA accumulation, the SL1(+) RNA from the 5` region of the PVX genome is also required for specific binding of PVX CP.