A restriction fragment map of Bacillus licheniformis temperate phage LP 52 DNA (molecular weight 38.5 X 10(6)) was established, using restriction endonucleases BamHI (8 target sites), BglI (10 sites,) BglII (13 sites) and EcoRI (22 sites). The map is linear, with well-defined ends, without any signs of circular permutation. The DNA of a related phage, LP 51, produced identical restriction fragments. At least 62% DNA of LP 52 has been found homologous to the DNA of the recently discovered, morphologically quite dissimilar, phage I, as demonstrated by hybridization of electrophoretically separated restriction fragments of DNA. Under the same conditions, the DNAs of LP 52 and of the morphologically similar Bacillus subtilis phage phi 105 did not cross-hybridize. The homologous regions in the genomes of phages LP 52 and I have been shown to be colinear. Comparison of the cleavage maps of phages LP 52 and I has shown that, within the regions of homology, not a single restriction fragment and few restriction sites have been conserved during divergent evolution. Three major regions of heterology were defined; the longest one, covering the right-hand end of the map (73 +/- 2.75% up to 100% LP 52 genome length) appeared to contain genes coding for structural proteins of the virions; a shorter region at the left-hand end of the map (coordinates zero to 10.3 +/- 3.3% LP 52 genome length) and a very short central region (coordinates 41.8-43.9%) could be identified, the latter apparently containing a regulatory locus responsible for the heteroimmune behavior of the two phages. Recombinants between phages LP 52 and I were isolated. Mapping of recombinant genomes has indicated mutual substitution of allelic pieces of LP 52 and I DNAs upon strict conservation of overall genome length.