Leptospira interrogans is a diverse species in which individual serovars have distinctive restriction fragment length polymorphisms that are useful in strain identification. Many of these polymorphisms can be detected using hybridization probes derived from insertion sequences; an observation that suggests these IS elements are active and can transpose in L. interrogans. Two spontaneous mutants of L. interrogans serovar Pomona strain RZ11 were isolated by immune selection and characterized. Changes in the size and antigenicity of LPS from these mutants were detected. Genetic analysis showed that both mutants have additional copies of an IS3-like element, designated IS1501, that are not present in the parental strain. One mutant, GT211, has a single additional copy of IS1501, whereas the other mutant, GT210 has three additional copies of IS1501 relative to strain RZ11. IS1501 transposition generated 3-bp direct repeats from target sequences flanking the insertion site. RT-PCR analysis of transcripts at altered loci showed IS1501 transcripts extended into adjacent sequences. These data are the first to show spontaneous transposition of an endogenous Leptospira insertion sequence, and suggest that IS1501 may be capable of gene activation.