Cells of 30 different strains of oral streptococci were grown in a chemically defined medium supplemented with [14C]glycerol to determine their ability to incorporate the labeled glycerol. Of the five species tested, only two, the rat-type strains (Streptococcus rattus) and strains isolated from wild rats (Streptococcus ferus), were able to incorporate the nonfermentable substrate, glycerol. For those strains capable of incorporating glycerol, the amount incorporated ranged from 0.15 to 0.43% of the cellular dry weight and followed simple saturation kinetics. The amount of glycerol incorporated depended solely on the concentration of glycerol in the growth medium. As a result, cultures exposed to low concentrations of glycerol ceased incorporation of the labeled glycerol before cessation of exponential growth.