The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii) diethyl-aminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at -80 degrees C in the presence of beta-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenedia-minetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NAD-dependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)-dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate and NAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K(m) values for mannitol-1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K(m) values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine 5'-triphosphate and fructose-6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.