beta-Galactosidase (beta-gal, EC 3.2.1.23) and beta-D-phosphogalactoside galactohydrolase (beta-Pgal) activities were observed in all of 13 Lactobacillus species studied except L. casei and L. buchneri. Only the latter enzyme was detected in nine strains of L. casei. The beta-gal from L. thermophilus and the beta-Pgal from L. casei were purified and characterized. In comparison with beta-gal, the beta-Pal was slightly less active (V(max) values were 28.9 and 50.0 mumoles per mg per min, respectively), but the substrate affinitives were similar (K(m) values were 1.69 x 10(-3) M and 1.59 x 10(-3) M, respectively). Although the two enzymes had similar amino acid compositions, the molecular weight of beta-gal was 5.4 x 10(5) and that of beta-Pgal was 1.3 x 10(5). The beta-gal from L. thermophilus and the beta-Pgal from L. casei had optimal temperature and pH activity values of 55 C at pH 6.2 and 37 C at pH 5.0, respectively. The complete absence of beta-gal from a homofermentative Lactobacillus species of industrial importance is further evidence of the heterogeneity of this genus.