Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45 degrees C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in beta-galactosidase (beta-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or beta-gal while B2 strains also lack a s-layer but do possess beta-gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have beta-gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in beta-gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.