Several synthetic approaches to N-alkylated derivatives of 4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid are described. The final compounds have been evaluated as potential inhibitors of the GABA transport proteins GAT-1 and GAT-3. The biological assays used were based on bovine material or porcine brain. As compared to the corresponding 4-unsubstituted compounds, the 4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid derivatives showed a significant decrease in the inhibitory potency at both GAT-1 and GAT-3 with only four compounds having reasonable affinity to GAT-1 (IC(50): 5.1, 6.6 and 9.4 microM) or GAT-3 (IC(50): 19.9 microM), respectively. The biological data of the 4-hydroxypyrrolidine-2-acetic acid derivatives indicates that (2S)-configuration at the C-2 position for potent inhibition of GAT-1 and (4R)-configuration at the C-4 position for potent inhibition of GAT-3 may be crucial.