Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and de®nitive DNA sequencebased identi®cation system for unknown isolates of the Lact. acidophilus complex. A I500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was ampli®ed from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was suf®ciently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and de®nitive method for the identi®cation of Lact. acidophilus complex members.