A detailed restriction endonuclease map for the genome of Bacillus subtilis phage SP01 is presented. Sites of cleavage for the restriction enzymes BglII, EcoRI, HaeIII, and SalI were determined. This physical map showed that SP01 DNA was 140 kilobases in length and contained a repeated sequence of 12.4 kilobases at its termini. Combined with previously published information, we were also able to identify the general locations of genes expressed at early, middle, or late times in the phage lytic cycle. In particular, early genes were largely clustered in the terminal repeats, whereas a major cluster of late genes was located in the left-central portion of the genome.