Two approaches to synthesize molecularly imprinted polymers with affinity for folic acid and other substituted pteridines have been compared. In the first approach, the folic acid analogue methotrexate was used as template and functional monomers capable of generating selective binding sites were searched in a miniaturized screening system based on binding assessment in the batch mode. Highest selectivity was seen using 2-vinylpyridine as functional monomer, which was confirmed in the chromatographic mode for a batch synthesized on a gram scale. However, the retentivity and selectivity of this phase were insufficient for anticipated applications. In a second approach, using methacrylic acid as the functional monomer organic soluble inhibitors for the enzyme dihydrofolate reductase; wars used to develop sites complementary toward the pteridine substructure. This resulted in materials showing enhanced selectivity for substituted pteridines when evaluated by HPLC. Thus, methotrexate and leucovorine were selectively retained in mobile phases of either low or high aqueous content, thus showing the typical bimodal retention behavior of previously reported MIPs. In organic mobile-phase systems, the inhibitor used as template had an influence on the retentivity and selectivity of the MIP. The polymer imprinted with trimethoprim retained all folic acid analogues strongly and showed the highest selectivity among the MIPs in an organic mobile-phase system. This was supported by Scatchard analysis resulting in biphasic plots and a quantitative yield of high-energy binding sites. All templates were shown to associate strongly with MAA in CDCl3 the strength of association correlating roughly with the template basicity and the selectivity observed in chromatography. Nonparallel complexation-induced shifts indicated formation of 1:2 template monomer complexes at concentrations corresponding to those of the prepolymerization solutions.