Promoter-active fragments were isolated from the genome of the probiotic organism Lactobacillus rhamnosus strain GG using the promoter-probe vector pNZ272. These promoter elements, together with a promoter fragment isolated from the vaginal strain Lactobacillus fermentum BR11 and two previously defined promoters (Lactococcus lactis and Lactobacillus acidophilus ATCC 4356 slpA), were introduced into three strains of Lactobacillus. Primer-extension analysis was used to map the transcriptional start site for each promoter. All promoter fragments tested were functional in each of the three lactobacilli and a purine residue was used to initiate transcription in most cases. The promoter elements encompassed a 52- to 1,140-fold range in promoter activity depending on the host strain. Lactobacillus promoters were further examined by surveying previously mapped sequences for conserved base positions. The Lactobacillus hexamer regions (-35: TTgaca and -10: TAtAAT) closely resembled those of Escherichia coli and Bacillus subtilis, with the highest degree of agreement at the -10 hexamer. The TG dinucleotide upstream of the -10 hexamer was conserved in 26% of Lactobacillus promoters studied, but conservation rates differed between species. The region upstream of the -35 hexamer of Lactobacillus promoters showed conservation with the bacterial UP element.