Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription. The combination of the s-y and s-z pairs provides a powerful tool to prepare both y-containing mRNA and s-containing tRNA for efficient coupled transcription-translation systems, in which the genetic code is expanded by the coclon-anticodon interactions mediated by the s-y pair. In addition, the nucleoside of s is strongly fluorescent, and thus the s-z pair enables the site-specific fluorescent labeling of RNA molecules. These unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.