A restriction endonuclease analysis of the hemolytic plasmid pSU316 has allowed location of the cleavage sites for the endonucleases BamHI, XbaI, KpnI, Bg/II, Sa/GI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pED100 have shown that the tra region of pSU316 lies in a segment comprising part of Sa/GI fragments S-1 and S-3 and the entire fragment S-4. The positions of other plasmids coded functions, namely the replication functions and alpha-hemolysin production, have been determined in the physical map.