Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na+/solute symporter family. Using the Na+/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the domain. G328, S332, Q345, and L346 were newly identified as important for Na+-coupled proline uptake. Placement of Cys at one of these positions altered Km(pro) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K0.5(Na+) (S332C, 13-fold decreased; Q345C, 19- fold increased), and/or Vmax [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane- permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na+ alone had no effect. Membrane-impermeant methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na+ but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na+ and/or proline translocation pathway of PutP.