In order to analyze mycotoxin sterigmatocystin (STC) by SERS spectroscopy, SERS substrates based on silver nanoparticles dispersed in a colloidal suspension and immobilized on a solid support were investigated. Freshly prepared silver colloid, concentrated colloid and colloid aggregated by addition of inorganic salts (calcium nitrate, sodium chloride) were used, while glass filter paper, glass treated with hydrofluoric acid and glass silanized with (3- aminopropyl)trimethoxysilane were employed as solid carriers for the deposition of silver nanoparticles. Silver nanoparticles were prepared by reducing silver(I) nitrate with trisodium citrate or hydroxylamine hydrochloride. Enhancement of Raman scattering of STC (1×10−5 mol dm−3) was not obtained with any of the investigated substrates, implying that the analyte molecules were not adsorbed on the silver nanoparticles surface regardless of the substrate type. Attractive electrostatic interactions between the neutral analyte molecules and the anion layer on the surface of the silver nanoparticles were not possible, nor was a chemical bond formed between the oxygen atoms of the analyte molecule and the surface of the silver nanoparticles. The SERS spectrum of aflatoxin B1 (1×10−5 mol dm−3) was also not observed.