The mechanism of renal transport of L- and D-serine by membrane vesicles prepared from either whole cortex, pars convoluta or pars recta of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Transport studies carried out with different salt gradients and by employing various ionophores showed that uptake of both L- and D-serine by luminal membrane vesicles from whole cortex was mediated by an Na+-dependent and electrogenic transport process. Eadie-Hofstee analysis of experimental data, obtained under extravesicular greater than intravesicular NaCl gradients, revealed the existence of multiple transport systems for L-serine but only one system for the D-isomer. The value of KA (the concentration producing a half-maximal optical response) for the D-serine transport system was calculated to be approximately 30 mM. Luminal membrane vesicles from pars convoluta take up both L- and D-serine by a single and common transport system. KA values for L- and D-serine transport were calculated to be 3.7 and 30 mM, respectively. Luminal membrane vesicles from pars recta take up L-serine by means of two transport systems, one of high affinity (KA = 0.37 mM) and the other of low affinity (KA = 10 mM). By contrast, no D-serine transport by these membrane vesicles could be detected. Uptake of L-serine by basolateral membrane vesicles is Na+ independent and electroneutral. Filtration studies showed that the transport is saturable (Km = 25-30 mM) and is inhibited by the presence of L-phenylalanine (but not by D-serine), indicating carrier-mediated uptake of L-serine.