Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.