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Abstract: Full Paper. Molecularly imprinted polymers (MIPs) for 17beta- estradiol were prepared following a multistep procedure including swelling and polymerization using polystyrene seeds, 4-vinylpyridine as the functional monomer and ethylene glycol dimethacrylate (EDMA) as crosslinker. Dibutylphthalate (DBF) and toluene were employed as swelling and porogenic compounds. All the samples showed a monomodal and very narrow diameter distributin with mean diameter values in the 2.0-4.7 mum range. The microspheres prepared in the presence of the template molecule present surface available vinyl pyridine unit content higher than those prepared without the template. The amphiphilic nature of the pre-polymerization complex between vinyl pyridine and 17beta-estradiol can be claimed,to be responsible for a preferential location on the vinyl pyridine units at the microsphere surface. The ability of molecular recognition depends on the accessibility of interaction sites located on surface of polymeric particles that increases when the ratios EDMA/polystyrene seed and DBF/polystyrene seed increase