The presence of two chromosomes makes Leptospira unusual amongst its closest relatives in the bacterial world. The Leptospira genome is in a state of flux, as indicated by the presence of many chromosomal rearrangements that alter genetic organization between individual serovars. It is therefore somewhat remarkable that at least two Leptospira loci (LPS biosynthetic genes and the S10-spc-alpha ribosomal protein operon) form large, extended operons that are among the longest bacterial operons reported to date. Insertion sequences (IS) that are distributed throughout Leptospira genomes contribute to the formation of rearrangements. These elements can transpose and disrupt the integrity of genes, or alternatively, can activate cryptic genes by providing promoter activity to genomic sequences downstream of the insertion site. Bioinformatics and experimental functional analyses were used to characterize the L. interrogans genomes and thus gain insight into this organism's biology. Quantitative analysis of the L. interrogans serovars Lai and Copenhageni genomes showed that these bacteria are proficient in environmental sensing and response, and in nutrient transport. These data support epidemiological evidence that L. interrogans is transmitted primarily by passage through environmental sources. Few pseudogenes were detected in either strain, suggesting that there is sufficient selective pressure to maintain a highly functional genome. However, several genes were identified that are complete in one strain but have frameshifts in the other that may affect phenotype. Further differences in phenotype may also result from gene acquisition, and we found several large, serovar-specific gene clusters. Analysis of an ECF locus from L. interrogans serovar Pomona is used to show how RT-PCR and expression vectors can be used to localize promoters in L. interrogans. Antisera produced against recombinant fusion proteins were used to detect invasion of lung, liver, and kidney during experimental infection of hamsters with serovar Pomona. These data are consistent with some of the clinical manifestations of severe leptospirosis and help to illustrate how genomic analysis can aid in the understanding of these pathogenic bacteria.