An acceptor stem G3.U70 base pair is a major determinant of the identity of an alanine transfer RNA. Hairpin helices and RNA duplexes consisting of complementary single strands are aminoacylated with alanine if they contain G3.U70. Chemical synthesis of RNA duplexes enabled the introduction of base analogs that tested the role of specific functional groups in the major and minor grooves of the RNA helix. The results of these experiments indicate that an unpaired guanine 2-amino group at a specific position in the minor groove of an RNA helix marks a molecule for aminoacylation with alanine.