ICEBs1 is an integrative and conjugative element (conjugative transposon) integrated into trnS-leu2 in Bacillus subtilis. In response to DNA damage or high concentrations of potential mating partners, ICEBs1 can excise and transfer to various recipients, including other species. We found that excision of ICEBs1 occurs by site-specific recombination within 60 bp direct repeats that mark the junctions between ICEBs1 and chromosomal DNA. Excision required two ICEBs1 genes, int (integrase, ydcL), predicted to encode a tyrosine recombinase similar to that of phage lambda, and xis (excisionase, sacV). Ectopic expression of xis was sufficient to induce excision of ICEBs1, indicating that regulation of xis transcription by DNA damage and peptide signalling normally controls excision. Int, but not Xis, was needed for site-specific integration. We found that in the absence of the primary bacterial attachment site (attB) in trnS-leu2, ICEBs1 integrated in secondary attachment sites that are similar to a 17 bp sequence in attB. In the absence of int, ICEBs1 could recombine into the chromosome by RecA-dependent homologous recombination, provided ICEBs1 contained a region of sequence identity to a chromosomal locus.