This paper reports a convenient method for immobilizing biologically active ligands to self-assembled monolayers of alkanethiolates on gold (SAMs). This methodology is based on monolayers that present maleimide and penta(ethylene glycol) groups. The maleimide groups react efficiently with thiol-terminated ligands, whereas the penta(ethylene glycol) groups prevent the nonspecific adsorption of protein to the substrate. The rate and selectivity of the immobilization of a ferrocene-thiol conjugate were characterized using cyclic voltammetry. This paper presents three examples of biochips prepared using this methodology. In the first example, four carbohydrate-thiol conjugates were immobilized to monolayers and the lectin-binding properties of the substrates were examined using fluorescence and surface plasmon resonance spectroscopy. The second biochip was used to study the enzymatic phosphorylation of the immobilized peptide IYGEFKKKC by the tyrosine kinase c-src. Monolayers presenting this peptide were then used to study the inhibition of the enzyme in an array format. The final class of substrates, which presents the tripeptide Arg-Gly-Asp, was used for studies of integrin-mediated cell adhesion. The immobilization methodology described here, which combines the structural order and inert properties of SAMs with the efficient reaction between soluble thiol and surface-bound maleimide groups, will be useful for preparing substrates for a wide range of applications in basic science and biotechnology.