RNA is known to interact with Mg(2+) when assuming higher-ordered tertiary configurations. Structurally, when tRNA molecules interact with Mg(2+), they consistently form a L-shape conformation each time they are synthesized. Therefore, if Mg(2+) can induce tertiary structure formation, then binding to alternative cations could produce alternative tertiary configurations. By utilizing circular dichroism and mobility gel-shift assays it was observed that tRNA structure can be altered when in the presence of different divalent cationic species. Formation of these alternative structural configurations was further validated by aminoacylating these tRNA structural anomalies with their native enzyme, which resulted in markedly different degrees of activity. Thus, it was confirmed that structural changes do occur when tRNA forms complexes with different cations. To better understand these structural changes, quantitative cation binding to tRNA was determined through titrations as well as ICP-OES analysis, which indicated that the metal ions can bind to the tRNA structure in specific and non-specific ways. Lastly, it was observed through stopped-flow kinetics that tRNA can associate/dissociate from different cations to varying degrees, thus forming cation-specific complexes at unique rates.