Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.