Acquisition of foreign DNA by horizontal gene transfer is seen as a major source of genetic diversity in prokaryotes. However, strongly divergent DNA is not genomically integrated by homologous recombination and would depend on illegitimate recombination (IR) events which are rare. We show that, by two mechanisms, during natural transformation of Acinetobacter baylyi two IR events can integrate DNA segments. One mechanism is double illegitimate recombination (DIR) acting in the absence of any homology (frequency: 7 x 10(-13) per cell). It occurs about 10(10)-fold less frequent than homologous transformation. The other mechanism is homology-facilitated double illegitimate recombination (HFDIR) being about 440-fold more frequent (3 x 10(-10) per cell) than DIR. HFDIR depends on a homologous sequence located between the IR sites and on recA(+). In HFDIR two IR events act on the same donor DNA molecule as shown by the joint inheritance of molecular DNA tags. While the IR events in HFDIR occurred at microhomologies, in DIR microhomologies were not used. The HFDIR phenomenon indicates that a temporal recA-dependent association of donor DNA at a homology in recipient DNA may facilitate two IR events on the 5' and 3' heterologous parts of the transforming DNA molecule.