We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.