A reevaluation of the specificity of system y+, the classical transporter for cationic amino acids is presented. System y+ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na+. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y+, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y+ in human erythrocytes with very low affinity. Inhibition constants (Kiy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na+, K+ or Li+ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y+ was shown to be clearly distinct from that of system y+L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na+ and which influenced the classical definition of system y+.