Transcription of yeast tRNA genes in vitro requires, in addition to RNA polymerase III, two accessory factors which are resolved by ion-exchange chromatography. One of these transcription factors (factor C) binds to tRNA genes. The stability of factor C-tRNA gene complexes is gene-dependent: the tRNAAGGArg gene forms a highly stable complex while tRNA3Leu and tRNATyr gene complexes are unstable under our standard assay conditions. To determine how differences in tRNA gene structure affect factor C binding, mutant tRNATyr genes, internally deleted tRNA3Leu genes and hybrid transcription units containing both tRNATyr and tRNA3Leu segments were compared in their abilities to stably bind factor C. Sequence changes in either of the two highly conserved promoter elements (A block and B block) affect factor C complex stability. Changes towards the consensus sequence increase complex stability while changes away from the consensus sequence drastically reduce stability. Also, the distance separating the A and B blocks affects complex stability; 34-53 bp gives highest stability. These results indicate that the stable binding of transcription factor C to tRNA genes involves interactions with both A block and B block sequences.