The interactions of two cystatins with a viral cysteine protease were studied using several types of assays. Complex formation between the protease and inhibitor was directly demonstrated using a gel retardation assay. It was also shown that the formation of enzyme inhibitor complexes could occur after first binding either the enzyme or the inhibitor to filter paper, and ultimately decorating the complex with antibody and radio-labelled protein A or by preparing one of the protein ligands with an internal radiolabel. The procedure can be adapted to provide a method for screening expression libraries for protease or inhibitor genes. The inhibition of a cysteine protease by a cystatin was shown not to directly involve binding to the active site thiol of the enzyme, but rather to be the result of a steric block in the active site region which prevents large affinity labels and protein substrates from reaching the active site.