The periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli transport sn-glycerol-3-phosphate and maltose, respectively. The UgpC and MalK proteins of these transport systems, which couple energy to the transport process by ATP-hydrolysis, are highly homologous, suggesting that they might be functionally exchangeable. Complementation experiments showed that UgpC expression could restore growth of a malK mutant on maltose as a carbon source, provided that it was expressed at a sufficiently high level in the absence of the integral inner membrane components UgpA and/or UgpE of the Ugp system. Conversely, MalK expression could complement ugpC mutants and restore the utilization of sn-glycerol-3-phosphate as a phosphate source. The hybrid transporters appeared to be less efficient than the wild-type systems. The complementation of ugpC mutations by MalK was strongly inhibited by the presence of glucose or alpha-methylglucoside, which are substrates of the phosphotransferase system. This inhibition is probably due to hypersensitivity of the hybrid UgpBAE-MalK transporter to inducer exclusion. UgpC expression did not complement the regulatory function of MalK in mal gene expression. The exchangeability of UgpC and MalK indicates that these proteins do not contribute to a substrate-binding site conferring substrate specificity to the transporter. These are the first examples of functional, hybrid periplasmic permeases in which the energy-coupling components could be functionally exchanged.