We constructed transformation of B. subtilis 168 which acquired genes for site-specific restriction endonucleases. These endonucleases originated from various strains of B. subtilis and were classified into five groups based on the specificity of the sequences recognized by the enzymes. We examined the loci of genes for site-specific restriction endonucleases belonging to different groups: hsrE determined Endo. R. Bsu1231 (I), hsrB Endo.R.Bsu1247(I), hsrR Endo.R.BsuR and hsrC Endo.R.Bsu-1247(II). One gene, hsrE, was located between sacA and purA by transduction crosses with phage PBS1, and another gene, hsrB, between hsrE and purA. Genes hsrR and hsrC had been suggested to be allelic or closely linked by previous studies with transformation. We located hsrR and hsrC between purB and tre. Our previous observation and this study show that B. subtilis 168 has at least three independent loci on the chromosome for four genes for site-specific restriction endonucleases in addition to the locus for the original restriction activity (Bsu168-specific restriction) of strain 168.