System A activity is a highly regulated mechanism for the active transport of zwitterionic amino acids into mammalian cells. Monoclonal antibodies generated against a previously unidentified rat liver plasma membrane-associated protein were shown to immunoprecipitate solubilized System A transport activity. The immunoreactive protein was later determined by immunoblotting and peptide microsequencing to be rat liver alpha-fodrin (non-erythroid spectrin). Antibody against ankyrin, a protein that often serves as a bridge between integral membrane proteins and fodrin, also immunoprecipitated System A transport activity. Fractionation of solubilized plasma membrane proteins on sucrose gradients revealed that the System A transporter co-migrated as a complex with fodrin and ankyrin, even in the presence of detergent and urea. In contrast, the System N amino acid transporter does not co-migrate with ankyrin and fodrin, nor does the anti-fodrin antibody immunoprecipitate System N activity. The present data are the first to demonstrate an association between an organic solute transporter and the membranocytoskeletal proteins ankyrin and fodrin.