Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclcfromPseudomonas knackmussiiB13 as model. By using self-transfer of ICEclcfrom strain B13 toPseudomonas putidaandCupriavidus necatoras recipients, we show that ICEclccan target a number of differenttRNAGlygenes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclcbased on theattRsequence, and we could show that ICEclcwill insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclcis excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, includingBordetella petri,Pseudomonas aeruginosaandBurkholderia.