Some species of Lactobacillus are of major industrial and health significance as fermenting agents in the manufacturing of food products, as food preservatives, as "probiotic" bacteria or as vaccine delivery vehicles. In spite of their importance, there is a paucity of published information on their genome organization and structure. In this study, a combination of pulsed field gel electrophoresis (PFGE) and hybridization approaches was used to investigate the genome of L, gasseri neotype strain ATCC33323. PFGE analysis of chromosomal DNA (after digestion with the rare-cutting restriction enzymes I-Ceu1, Csp1, Sma1, Apa1, and SgrA1) allowed the chromosome size of L. gasseri to be estimated at 1.96 Mbp, and also revealed the presence of a linear plasmid of 48.5 kbp. A physical map of the L. gasseri chromosome, containing 6 sites for the enzymes I-Ceu1 and 12 for Csp1, was constructed. Placed on the map were the genes dnaA and gyrB (usually located close to the origin of replication on the bacterial chromosome) and 18 ribosomal RNA (rrn) genes. Mapping analysis also revealed that the chromosome contained six rrn operons, and that one of them was inverted in orientation with respect to the others. Each rrn operon contained a single copy of each of the three rrn genes, 23S rRNA (rrl), 16S rRNA (rrs) and 55 rRNA (rrf) gene. The constructed physical map should be a useful foundation forgenomic and genetic studies of the lactobacilli and provides a platform for applied research, such as the engineering of Lactobacillus strains with improved characteristics for industrial and probiotic applications.