In Salmonella typhimurium and Escherichia coli the high-affinity galactose transport system, which contains a periplasmic galactose-binding protein as an essential component, is encoded by the mgl genes. The entire mgl region of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment, which has been cloned into plasmid vectors. We determined the extent of the mgl region on this fragment by Tn5 mutagenesis, examination of lacZ fusions to mgl genes, and subcloning smaller restriction fragments. Polyacrylamide gel electrophoresis of protein preparations derived from strains carrying different plasmids was used to identify the mgl gene products. We conclude that the mgl operon consists of four genes that form a single transcription unit: mglB, mglA, mglE, and mglC. The mglB gene codes for galactose-binding protein (33,000 daltons), mglA codes for a membrane-bound protein of 51,000 daltons, and mglC codes for a 29,000-dalton membrane protein. The mglE product was less well characterized. Its existence was inferred from a mglE-lacZ protein fusion located between mglA and mglC. In addition, the coupled transcription-translation in vitro system indicated that mglE codes for a 21,000-dalton protein.