Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.