Sequence homology between the DNA molecules of the two temperate Bacillus subtilis bacteriophages, SPO2 and φ105, has been mapped by electron microscope observation of heteroduplexes. There is a region of partial homology covering about 14% of the genome (3.8 × 106 daltons) close to the center of the heteroduplex molecule, flanked by completely non-homologous regions of lengths about 1.2 × 107 and 1.0 × 107 daltons on the two sides. Within the central homologous region there is a characteristic pattern of duplex regions and single-strand loops. The amount of duplex decreases as the denaturing power of the solvent used for preparing the electron microscope grids increases; this indicates that the DNA molecules of the two phages are only partially homologous within the homology region. The heteroduplex patterns show that there are no completely homologous nor completely non-homologous gene size sequences within the central region of partial homology. Since the phages are serologically related, we conclude that the antigenic determinants for serological cross-reactivity in the phage tails are coded for by genes in the central region of homology. This conclusion is consistent with available genetic data. Comparison of genetic and physical data indicates that the genes for DNA synthesis and for clear plaque formation in the two phages are non-homologous. The molecular weights of SPO2 DNA and φ105 DNA are both calculated as 26.3 (± 0.3) × 106 daltons, from length measurements relative to φX174 RFII DNA. Both DNA's have cohesive ends and are capable of reversible cyclization; the joined ends dissociate more readily than do those of λ DNA. Our physical studies show that each phage DNA consists of a unique linear sequence and is not circularly permuted, in agreement with the conclusion from genetic studies that both phage maps are linear. © 1972.