The internal control elements of Neurospora crassa 5 S genes include an A box and a C box as in Xenopus and Saccharomyces cerevisiae, plus a novel element, the Ribo box at position +18 to +34. The Ribo box is also found in the 40 S rRNA promoter and a ribosomal protein gene but is absent from tRNA genes in N. crassa. The 5 S A box diverges from the tRNA A box consensus at two positions. We tested whether replacement of the 5 S A box with a tRNALeu A box sequence would increase 5 S gene transcription in vitro or would remove the requirement for the Ribo box. The 5 S gene with the tRNALeu A box was transcribed poorly, and the Ribo box and the C box are still required for transcription. We tested the function of the Ribo box and 5 S A box in a tRNA-like transcription unit by constructing hybrids between a 5 S gene and a tRNALeu gene. In the tRNA-like context, the 5 S A box supported a lower level of transcription than the tRNA A box, and the Ribo box was not required at all. Therefore, in N. crassa, all of the 5 S internal control elements are gene-specific. In particular, the 5 S and tRNA A box sequences are not functionally interchangeable and may bind different transcription factors. Transcription of the hybrids was initiated at the 5 S initiation site, suggesting that the mechanism of initiation site selection is the same in the 5 S and tRNA genes. Competition experiments with the tRNA B box suggested that the N. crassa 5 S and tRNA genes require at least one common transcription factor such as TFIIIC.