Uptake of L-proline, hydroxy-L-proline and 5-oxo-L-proline by luminal-membrane vesicles isolated either from whole cortex or from pars convoluta or pars recta of proximal tubules was studied by a spectrophotometric method. Uptake of L-proline and hydroxy-L-proline by vesicles from whole cortex was mediated by both Na+-dependent and Na+-independent, but electrogenic, processes, whereas transport of 5-oxo-L-proline in these vesicles was strictly Na+-dependent. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in luminal-membrane vesicles from whole renal cortex for the uptake of all these amino acids. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and from pars recta. In pars recta transport of all three amino acids was strictly dependent on Na+ and occurred via a high-affinity system (half-saturation: 0.1-0.3 mM). Cation-dependent but Na+-unspecific transport of low affinity for L-proline and hydroxy-L-proline was exclusively localized to the pars convoluta, which also contained a Na+-preferring system of intermediate affinity (half-saturation: L-proline, 0.75 mM; hydroxy-L-proline, 1.3 mM). 5-Oxo-L-proline was transported by low-affinity and Na+-dependent systems in both pars convoluta and pars recta. Competition experiments revealed that transport systems for L-proline and hydroxy-L-proline are common, but indicated separate high-affinity transport systems for 5-oxo-L-proline and L-proline in luminal-membrane vesicles from pars recta. The physiological importance of the presence of various neutral amino acid-transport systems in different segments of the proximal tubule is discussed.