Overoxidized polypyrrole colloids imprinted with L-lactate were prepared to evaluate the performance of the overoxidation pseudo-template technique developed by the authors. A polypyrrole colloid that had been prepared from a mixture of monomer (pyrrole), dopant (L-lactate), steric stabilizer (poly(vinylpyrrolidone)) and oxidizing agent (peroxodisulfate) was electrochemically overoxidized at +1.5 V vs Ag/AgCl to create a complementary cavity for recognition of the molecules, which were structurally similar to dopant, through dedoping. As a target molecule for enantioselective uptake into the overoxidized colloid, we selected alanine, which is structurally different from the template (lactate) only in one side chain (alanine, -NH2; lactic acid, -OH). The overoxidized polypyrrole colloid showed higher affinity for L-alanine than for D-alanine, and an uptake ratio (L/D) of as high as 11 +/- 4 was observed under optimum conditions. Uptake reached equilibrium in 10 min, thanks to the high surface area and short diffusion length in the colloidal particle. Further, to confirm the complementarity of the cavity, the effect of side chain size on uptake of several alpha-amino acids was examined to indicate that the uptake amount decreased with increasing molecular volume of the L-amino acids.