The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2Apro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2Apro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2Apro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2Apro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2Apro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2Apro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2Apro, suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.