The bacteriophage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), forms complexes with specific sites in SPO1 DNA. We have investigated the binding of TF1 to one of its preferred sites in which the normal 5-hydroxymethyluracil (hmUra) of SPO1 DNA has been replaced by thymine and have also investigated the binding of a bacterial Type II DNA-binding protein (from Bacillus stearothermophilus) to the hmUra- and thymine-containing forms of the same DNA segment. Our results show that TF1 binds selectively to this high affinity binding site only in hmUra-containing DNA and that the bacterial Type II DNA-binding protein interacts nonspecifically with both forms of DNA.