The 5S gene-specific transcription factor TFIIIA forms an exceptionally stable complex with the internal promoter of the 5S RNA gene. Approximately 1 to 5% of TFIIIA-DNA or deoxyoligonucleotide complexes are stable to harsh denaturation conditions and can be resolved by electrophoresis in the presence of SDS. These complexes are resistant to acidic conditions (0.1 N HCl, 55 degrees C, 2h) suggesting that the interaction may be through a covalent bond. Complex formation does not result in DNA strand scission and studies of the chemical sensitivity of the complex suggest that the TFIIIA-DNA linkage may be through a phosphoramidate bond. Covalent complexes are formed with both the noncoding (RNA-like) and coding strands of the internal control region. The predominant sites of TFIIIA-DNA adducts have been mapped to the 3' end of the 5S gene internal control region, the region previously shown to exhibit essential guanine and phosphate contacts with TFIIIA.