Band 3, the anion transporter, is a ubiquitous protein. It is present in brain and all other tissues. Not only is band 3 present in cell membranes, but also in nuclear, Golgi, and mitochondrion membranes. Band 3 is involved in respiration, acid-base balance, and is the major structural protein linking the plasma membrane to the cytoskeleton. Thus, alterations/mutations in the transport segment of the band-3 molecule might be expected to be of major importance. We discovered and sequenced a mutation of band 3, high-transport band 3 (HTbd3), that exhibits anion transport that is 2-3 time above normal. Anion transport studies of the family members revealed that red cells from the proposita, one of two siblings, and both parents had abnormally increased anion transport (increased Vmax). We used synthetic peptides of band 3 to help localize the change along the band-3 molecule. Results suggest that high-transport band 3 is altered in or near residue 869-883. This places the alteration toward the carboxyl terminal of band 3. cDNA sequencing demonstrated that the mutation was a proline to leucine at residue 868. A peptide was synthesized corresponding to residues 853-870 for testing in the anion transport inhibition assay. This peptide significantly inhibited anion transport (p < or = 0.001) indicating that it is an anion transport/binding region of band 3. Thus, DNA technology confirms the validity of the anion transport inhibition assay for localizing transport regions. Glucose transport is decreased in affected individuals. The HTbd3 mutation appears benign as determined by the red cell aging panel. IgG binding, creatinine, and glyceraldehyde-3-phosphate dehydrogenase are normal. Our studies indicate that the most rapid and sensitive techniques for detecting band-3 alterations are polyacrylamide gel electrophoresis, IgG binding, and anion transport studies. This is the only mutation of band 3 discovered to date the affects the transmembrane, anion transport region of band 3.