MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in Escherichia coli. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L₃₂₃Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.