All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.