Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymatic activity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of trypsin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permit analysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDS-PAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretreatment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.