The proU locus of Salmonella typhimurium encodes an osmotically induced betaine transport system. We have identified a 31 kDa periplasmic protein, encoded by proU, whose synthesis is induced by osmotic stress. A specific betaine-binding activity with a KD of about 1 microM is also present in the periplasm of osmotically induced cells. This activity is absent in those proU mutants which lack the 31 kDa periplasmic protein. Thus, ProU is a periplasmic binding-protein-dependent transport system.