The immobilized metal ion affinity (IMA) interaction of different serine proteases, namely porcine and bovine trypsins and BPN' and Carlsberg subtilisins, was studied on Sepharose-IDA-CuII. Both trypsins were resolved into their different subspecies, whereas the subtilisins appeared as only one species. The use of diethyl pyrocarbonate-modified enzymes demonstrated the contribution of histidine(s) as the sole interacting site(s). The use of different peptidic and chemical inhibitors complexed to the enzymes confirmed the contribution of histidine(s) as the interacting site(s) and further resulted in different chromatographic patterns for the free and complexed serine proteases. Comparison of the chromatographic data for each enzyme with the accessible surface area calculation by molecular modelling on the available crystallographic structure allowed us to hypothesize a map of the surface-accessible histidine on each enzyme.