Abstract We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb. To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20–30 kb inserted DNA. Subsequently, a cosmid walking strategy, combined with BamHI or EcoRI restriction analysis and hybridization, was used to construct the restriction maps for both enzymes. On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, accounting for a total size of approximately 180 kb. We also analyzed several rearranged derivatives of pMOL28 that were obtained after a process of temperature-induced mortality and mutagenesis (TIMM), which is characteristic for Alcaligenes eutrophusCH34 and related strains. The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivative of pMOL28 obtained after TIMM, were constructed. By comparing the pMOL28 and pMOL50 maps, at least two transposable elements were identified which participated in the formation of pMOL50 from pMOL28 during TIMM. These transposable elements were IS 1086,which was recently sequenced, and a new element named IS 1089,which is located on the 44-kb inserted DNA fragment in pMOL50. Partial sequencing of IS 1089revealed similarity of this element with IS 1071of the chlorobenzoate catabolic transposon Tn 5271of Alcaligenessp. BR60.