Promoter-like sequences from the chromosomal DNA of thermophilic strain Lactobacillus acidophilus ATCC 4356 were cloned. Analysis of the three DNA fragments showing promoter activity, designated P3, P6, and P15, were performed in Lactobacillus reuteri, Lactococcus lactis, and E. coli. The reporter cat-86 gene was expressed in all three bacterial species under control of the fragments P3 and P6. Fragment P15 showed promoter activity only in Lactobacillus reuteri and E. coli but not in Lactococcus lactis. The three host-specific transcriptional start points (TSPs) were used when transcription of the cat-86 gene was controlled by fragment P3 in Lactobacillus reuteri, E. coli, and Lactococcus lactis. Similarly, fragment P15 initiated transcription of the cat-86 gene at two distinctive sites in Lactobacillus reuteri and E. coli. Only within fragment P6, a common TSP was used in Lactobacillus reuteri and E. coli, but different from that used in Lactococcus lactis. Each TSP was preceded by the putative -35 and -10 hexamers. Computer analysis of the fragment P3 sequence revealed the existence of divergent promoter-like sequence (P3rev) located on the complementary DNA strand. Fragments P6 and P15 were also functional in Lactobacillus acidophilus ATCC 4356 from which chromosomal DNA they were originally cloned.