Abstract Lactobacillus acidophilus LTF42 metabolizes lactose by the phosphoenolpyruvate-dependent phosphotransferase system for lactose uptake and phospho-β-galactosidase (P-β-gal) that cleaves the lactose-6-phosphate formed. Strain LTF42 was found to carry a single 57-kb plasmid, pLA421. Since the Lac − strain LTF42-1, a pLA421-cured derivative of LTF42, has lost P-β-gal activity, pLA421 is likely to encode the gene for P-β-gal. A 4.8-kb SalI fragment of pLA421 containing the P-β-gal gene, designated pbg, was shotgun cloned in Escherichia coli. Further subcloning and deletion of this 4.8-kb SalI fragment allowed localization of the pbg gene on a minimal 1.9-kb ClaI- KpnI region. Analysis of the nucleotide sequence showed that the pbg gene was composed of 1,419 bp (473 amino acid residues) which can encode a protein with a molecular weight of 54,008. The deduced amino acid sequence of the pbg gene showed a degree of homology exceeding 60% with the P-β-gal amino acid sequences of other Gram-positive bacteria. Transformants carrying the recombinant plasmid pULA5EP containing the pbg gene on pLA421 expressed P-β-gal activities comparable to those of the parent strains.