The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties. The active form of repressor appears to be a tetramer. DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF). Three of these sites had been identified earlier as functional operators by genetic analysis. They share a common 14-base pair, asymmetric "core" sequence, 5'-GACGGAAATACAAG-3', termed OR. The three additional sites show significant homology with OR. For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix. Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model. However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction.