Spontaneous, phenotypically stable mutations at the beta-galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 bp long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1% identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4-21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.