A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.