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