Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown. To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis. The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymatic activities. Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming. In retrohoming, the intron reverse splices into a cognate intronless DNA site. Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA. Here we report the first experimental demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism. This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets. These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.