The aminoacylation of tRNA, a process in which amino acids first encounter RNA, may be closely related to the origin of L-amino acid homochirality in biological systems. A clear preference for L-amino acids as opposed to D-amino acids was noted in the efficient nonenzymatic aminoacylation of an RNA minihelix (progenitor of the modern tRNA) by an aminoacyl phosphate oligonucleotide. The steric clash between the side chains of D-amino acid and the terminal adenosine of the minihelix is the stereochemical mechanism underlying the chiral selectivity of the aminoacylation. Subtle structural changes dependent on sugar puckering could affect the positioning of the amino acids and hence the chiral selectivity. These results clearly suggest that tRNA aminoacylation may have been a critical step in determining L-amino acid homochirality.