A shuttle plasmid for Acinetobacter calcoaceticus and Escherichia coli has been constructed from a cryptic A. calcoaceticus lwoffi plasmid and pBR322. It is transformed to A. calcoaceticus BD413 by natural competency, yielding about 10(6) transformants per microgram of plasmid DNA. The ApR and TcR genes of pBR322 are functional in A. calcoaceticus. A gene bank was constructed from chromosomal A. calcoaceticus DNA and the shuttle plasmid. Direct transformation to A. calcoaceticus yielded about 95% recombinants, indicating a sixfold enrichment of recombinant plasmids compared to E. coli. One clone complementing a trpE mutation carried a 20-kb insertion and transformed with a 30-fold higher efficiency when compared to the vector. A deletion analysis of the shuttle plasmid indicates that 2.2 kb is necessary for autonomous replication and stable maintenance in A. calcoaceticus. No rearrangements of the DNA or loss of plasmids are found in that organism, even in the absence of selective pressure, when this sequence is present. A further insertional inactivation analysis creating lacZ transcriptional fusions suggests that the origin of replication (ori) is contained within about 1350 bp. Analysis of beta-galactosidase production in A. calcoaceticus indicates that only a weak promoter activity is directed out of one end of this ori. Its sequence contains A + T-rich regions, an 18-bp element with nearly perfect palindromic symmetry and eleven repeats of the consensus sequence, AAAAAATAT, eight of which are clustered within 360 bp. However, no open reading frames or significant homologies to other ori were found.