Recently, many Amyotrophic lateral sclerosis (ALS)-causing proteins reportedly undergo liquid-liquid phase separation (LLPS). It has also been shown that arginine-rich dipeptide repeat proteins (R-DPRs), poly(PR) and poly(GR), expressed from mutant C9ORF72 are prone to phase separation, and associate with membrane-less organelles formed by LLPS, disturbing their functions. Although R-DPRs share many biochemical features, such as the alternating of Arg, their subcellular localization and toxicity mechanisms are different. However, the mechanisms underlying these differences are unknown. In this study, we analyzed localization, intermolecular interactions, and LLPS of R-DPR variants, and found that these differences are determined by the degree of segregation of Arg charges (Miyagi et al., iScience 2023). In poly(PR), the segregation of arginine side chains by Pro is a critical factor in promoting nucleolar incorporation. In addition, Pro allowed interactions with molecules in a weak but highly multivalent manner. Still, Gly was incapable of separating the Arg well, so poly(GR) showed static localization in the cytosol. Poly(GR) binds to molecules in a strong, but less multivalent manner than poly(PR). These data reveal critical factors that determine biochemical differences in the R-DPRs.