Spectrophotometric assays of β–galactosidase (EC 188.8.131.52) and phospho-β–galactosidase (EC 184.108.40.206) activity were used to survey the lactose utilization pathways of lactic acid bacteria and bifidobacteria. β–Galactosidase activity was found in all six genera represented (Lactococcus, Streptococcus, Leuconostoc, Lactobacillus, Pediococcus and Bifidobacterium) while phospho-β–galactosidase was restricted to the lactococci, two Lactobacillus and two Leuconostoc species. A number of strains of Lactococcus lactis, Lactobacillus casei and Leuconostoc spp. contained both enzymes. Enzyme activities varied when cells were grown on different sugars, but in general were low or absent for cells grown on glucose compared with lactose. Two lactose-related compounds, lactulose and galactosyl lactose, believed to be specific growth factors for bifidobacteria, supported growth amongst a wide range of lactic acid bacteria in addition to bifidobacteria. Growth on galactosyl lactose was restricted to some but not all strains containing β–galactosidase, implying that the presence of β–galactosidase is insufficient by itself to ensure utilization of galactosyl lactose. DNA fragments that encoded the Lactococcus lactis subsp. cremoris phospho-β–galactosidase gene or the β–galactosidase genes of Streptococcus salivarius subsp. thermophilus or Lactobacillus delbrueckii subsp. bulgaricus were isolated and used as probes in DNA-DNA hybridizations. Little or no hybridization was detected between these probes and plasmid or genomic DNA isolated from heterologous species, despite the presence of the corresponding enzyme activity in the strains probed.