Twenty-nine different SUP4-o tRNATyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacing a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the T psi CG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.