Acquisition of new genetic information by horizontal gene transfer is a major mechanism of genetic adaptation and evolution in prokaryotes. Naturally transformable cells of Acinetobacter sp. were exposed to plant DNA from leaf and root tissue of transplastomic tobacco. With the aadA gene (resistance against spectinomycin and streptomycin) as anchor sequence, the transfer of segments of the tobacco plastid DNA to Acinetobacter by homology-facilitated illegitimate recombination occurred at a frequency of 1.2 x 10(-7) per cell, which was about 0.1% of the frequency of fully homologous transfers. Without anchor sequence, transfer was not detected (</=1.3 x 10(-10)). The integrated plastid DNA segments extended up to 2539 nucleotides and often encompassed tobacco genes (trnL, ycf5). Expression of trnL (leucyl-tRNA) in a transformant was shown by reverse transcription polymerase chain reaction. About 44% of integration events occurred at a single hot-spot and 38% at other multiply used sites. All illegitimate recombination sites were GC-rich microhomologies of 3-6 bp often neighboured by further microhomologies. The sites were located in plant DNA at the ends of distinct larger high-GC regions, which suggests a role for GC-aided association of heterologous sequences in illegitimate DNA end joining. The results show that integration of plant DNA into a bacterial genome by natural transformation is possible and is probably stimulated by hot-spots of illegitimate recombination.