Two modular elements (P5abc and Delta P5) in the Tetrahymena group I ribozyme can be separated physically to generate a two-piece ribozyme derivative consisting of a separately prepared P5abc (P5 RNA) and the rest of the intron (Delta P5 RNA). Molecular recognition in the interface assembling P5 RNA and Delta P5 RNA is strong and specific, and the catalytic ability of the two-piece ribozyme is comparable to that of the parent unimolecular ribozyme. We designed alternative P14 (L5c-L2) interacting modules participating in the assembly of P5 and Delta P5 and investigated their ability in the context of complex formation of the two-piece ribozyme and in vivo splicing of the unimolecular intron ribozyme. Combined use of alternative P14 and L5b-P6 interacting modules provided robust orthogonality to the P5/Delta P5 assembly interface of the bimolecular complex. (c) 2013, The Society for Biotechnology, Japan. All rights reserved.