Enterococci mainly from laboratory collections were separated by established and new or modified tests into two distinct species, S. faecalis and S. faecium. Of the established tests only reducing activity and potassium tellurite tolerance completely differentiated the organisms. Of the additional tests, the most useful were fermentation of glycerol in a soft agar medium containing fumarate, H2O2 formation from polyhydroxy alcohols and the ability to ferment them, H2O2 formation on a basal medium containing no added specific energy source, citrate fermentation and dissimilation of malate in the presence of glucose. Catalase activity and the ability to use malate as an energy source were also useful tests but did not completely differentiate the organisms.