Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P). G38P specifically binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome. One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin (oriL). The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR. Both sites contain two types of repeated elements (termed Box AB and A + T-rich region). The K(app) for the G38P-oriL DNA and G38P-oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively. G38P binds to the distant oriL and oriR sites cooperatively. DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A + T-rich region. Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through protein-protein interaction. G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it. These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted.