This paper describes a procedure for preparing mixed self-assembled monolayers (mixed SAMs) on gold that resist the nonspecific adsorption of proteins from solution. This method was tested using alpha-amino derivatives of omega-hydroxy- and omega-methoxy-oligo(ethylene glycols): H2N(CH2CH2O)(n)CH3 and H2N(CH2CH2O)(n)H (n = 3, 6). Mixed SAMs were prepared by all owing these amines to react with a SAM presenting interchain carboxylic anhydride groups. The resistance of the resulting surfaces to adsorption of several proteins-carbonic anhydrase (EC 4.2.1.1), ribonuclease A (EC 3.1.27.5), lysozyme (EC 3.2.1.17), and fibrinogen-was examined using surface plasmon resonance (SPR) spectroscopy. These mixed SAMs resist the nonspecific adsorption of proteins approximately as effectively as single-component SAMs prepared using the conventional method involving chemisorption of oligo(ethylene glycol)-terminated alkanethiols on gold. Characterization of the mixed SAM that presents a 1:1 mixture of -OCNH(CH2CH2O)(6)CH3 and CO2H/CO2-groups by polarized infrared external reflectance spectroscopy indicates that the ethylene glycol units are in an amorphous conformation. A model surface for use in studies of biospecific adsorption was synthesized by reacting the anhydride groups with a mixture of H2N(CH2CH2O)(6)H and H2N(CH2CH2O)(6)CH2CONH(CH2)(6)NHCOC6H4SO2NH2; the resulting system was examined for its ability to bind bovine carbonic anhydrase by SPR. The values of the relevant constants were k(off)= 0.0054 s(-1),k(on) = 13 000 M-1 s(-1), and K-d = 0.42 mu M. These values agree with values obtained by other means. The reaction of amines with SAMs that present interchain carboxylic anhydrides provides an experimentally simple route to the formation of mixed SAMs that resist the nonspecific adsorption of proteins or that adsorb a protein of interest biospecifically.