Proteins that bind to specific sequences in replication origins have been shown to be important components of both prokaryotic and eukaryotic DNA replication systems. An origin of DNA replication is located within the 5[superscript]' non-transcribed spacer region of the rRNA genes (rDNA) of Tetrahymena thermophila. Previous work has shown that several cis-acting mutations responsible for rDNA replication defects lie in upstream repeats of a transcriptional promoter element, the Type I repeat;Using FE(II)EDTA cleavage footprinting and gel-mobility shift assays, I identified and characterized two distinct DNA-binding activities that interact with the Type I repeat. One of these activities, designated ds-TIBF, binds preferentially to duplex DNA whereas the other, ssA-TIBF, binds preferentially to single-stranded DNA. Competition studies revealed that although ds-TIBF binds with high affinity to Type I sequences, it has only moderate sequence specificity but binds with high affinity to other duplex oligonucleotides containing dA/T stretches of greater than 6 nucleotides. In contrast, ssA-TIBF does not bind to generally A-rich oligonucleotides, but specifically interacts with single-stranded oligonucleotides corresponding to the Type I repeat;UV crosslinking of the partially purified protein to A-rich strand oligonucleotides followed by SDS gel electrophoresis demonstrated the binding activity of ssA-TIBF is associated with a polypeptide of approximately 95 kD. The native size of the binding activity determined by gel filtration chromatography is 205 kD, suggesting that ssA-TIBF may exist as a dimer or that the 95 kD polypeptide associates with another protein(s) in the cell;The nucleotide sequence requirements for optimal binding of ssA-TIBF were characterized. A deletion that removes part of one copy of the ssA-TIBF recognition sequence in the replication origin region has been shown to cause an rDNA replication defect in vivo. The affinity of ssA-TIBF for oligonucleotides reflecting this deletion is reduced by approximately 60% in vitro. Another copy of the Type I repeat, located only 20 nucleotides upstream from the transcription start site, is an essential component of the rRNA gene promoter. This element, although slightly different in sequence, is bound by ssA-TIBF with an affinity approximately equal to that of the Type I element in the replication origin region. These findings suggest that ssA-TIBF could be a transcription factor that also plays a role in rDNA replication.