Oligonucleotide mutagenesis was used to isolate mutations in membrane- spanning segments 6, 7, and 8 of MalF. MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli. The current structural model predicts eight transmembrane domains for MalF. Membrane-spanning segments 6, 7, and 8 of MalF flank or are part of the EAA-X3-G-X9-I-X-LP consensus region present in the cytoplasmic membrane subunits of the bacterial ABC transporter superfamily members. Mutations with two novel phenotypes with respect to substrate specificity of the maltose transport system were isolated. One mutant grew on minimal maltose media but not on media containing either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative. The other class of mutations led to a maltose minus but maltoheptaose plus phenotype. Nine of the isolated mutations leading to changes in substrate specificity were tightly clustered on one face of the postulated transmembrane helix 6. A similar clustering of mutations was detected in transmembrane domain 7. The majority of mutations in membrane-spanning segment 7 led to a protease-sensitive or a conditional phenotype with respect to MalF function or both. Mutations in transmembrane domain 8 appeared to be more randomly distributed. The majority of mutations in membrane-spanning segment 8 caused a Mal+ Dex- phenotype. Six Mal+ suppressor mutations isolated to two mutations in transmembrane domain 7 changed amino acid residues in membrane-spanning segment 6 or 8.