A biochemical and genetic analysis of d-mannitol metabolism in Salmonella typhimurium indicates that d-mannitol is phosphorylated by the phosphoenolpyruvate-dependent phosphotransferase system. d-Mannitol-1-phosphate is converted to d-fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. Two classes of mannitol mutants are described. Both map at about 115 min on the Salmonella chromosome. Mutants missing mannitol-1-phosphate dehydrogenase activity are mannitol sensitive; i.e., either growth is inhibited or the cells are lysed in the presence of mannitol. In a strain missing adenyl cyclase activity, the mannitol genes require exogenous cyclic adenosine-3',5'-monophosphate for expression.