Many designated substrates and inhibitors have been widely used to investigate the roles of caspases in apoptotic death during mammalian cell Culture. However, the specificities of these substrates and inhibitors have not been systematically evaluated. As a result, conclusions on the roles of specific caspases in apoptotic cells have been published inaccurately. In this study, the interaction between seven commercially available human caspases and their designated substrates and inhibitors was studied, Ac-YVAD-pNA, the designated substrate for caspase-1, is found to be the most specific substrate. All other substrates tested demonstrate cross-reactivity with several caspases. In relation to the enzyme, Caspase-2 is the most specific caspase, followed by caspase-9 and -6. Caspase-3 and -7 cleave three Substrates efficiently. The designated substrates for capsase-1 and -8 are not even their best Substrates. Fluoromethylketone (fmk) inhibitors exhibit no specificity towards different caspases even at low concentrations. In contrast, aldehyde inhibitor potency shows a distinct relationship to pNA substrate cleavage. These results show that some commonly used caspase Substrates and inhibitors lack the specificity required to monitor individual caspase activity. (C) 2008 Elsevier Inc. All rights reserved.