The first comprehensive comparative analysis of lactobacilli was done by comparing the genomes of Lactobacillus plantarum (3.3 Mb) and Lactobacillus johnsonii (2.0 Mb). L. johnsonii is predominantly found in the gastrointestinal tract, while L. plantarum is also found on plants and plant-derived material, and is used in a variety of industrial fermentations. The L. plantarum and L. johnsonii chromosomes have only 28 regions with conservation of gene order, totalling about 0.75 Mb; these regions are not co-linear, indicating major chromosomal rearrangements. Metabolic reconstruction indicates many differences between L. johnsonii and L. plantarum: numerous enzymes involved in sugar metabolism and in biosynthesis of amino acids, nucleotides, fatty acids and cofactors are lacking in L. johnsonii. Major differences were seen in the number and types of putative extracellular proteins, which are of interest because of their possible role in host-microbe interactions. The differences between L. plantarum and L. johnsonii, both in genome organization and gene content, are exceptionally large for two bacteria of the same genus, emphasizing the difficulty in taxonomic classification of lactobacilli.