The natural transformability of the soil bacterium Acinetobacter sp. ADPl (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 378021 91 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease Notl. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered Not1 cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of Notl, Ascl and I-Ceul recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColEl-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADPI.