It is difficult to modify the 3'-end of RNA transcribed in vitro from recombinant plasmid or phagemid DNA because of the need to retain a restriction endonuclease recognition site at the downstream end of the template in order to linearize the DNA before transcription. To avoid limitations on the 3'-sequence of RNA transcripts, we have modified a template-containing phagemid by inserting a FokI site outside the RNA gene sequence, positioned to cleave the template DNA to yield the desired 3' terminus. We demonstrate that this phagemid is an efficient template for tRNA transcription and use it to prepare mutants of Escherichia coli tRNA(Val) with modifications at the 3'-CCA end. Phagemids containing a FokI site should be suitable for in vitro transcription of large quantities of RNA of any length and with an unlimited variety of 3'-terminal sequences.