Introduction and Aims: Apoptosis contributes to the pathogenesis of hypoxic-ischemic brain injury. The caspases are the family of cysteine proteases which are pivotal in apoptosis. Several viral genes known to block apoptosis function as competitive inhibitors of these caspases. P35 (from autoradiographica california) and Cytokine response modifier (crmA) from the cowpox virus both contain regions which act as pseudosubstrates for some of the caspases. The specificity of these genes depends on the amino acid sequence (DQMD in p35 and LVAD in crmA) which mimics the preferred cleavage sites of the normal substrates of the caspase. Methods: In order to determine the important caspases in response to hypoxic neuronal injury, we mutated the pseudosubstrate region of crmA to match the predicted substrate preferences of the different caspase groups1. Using both stable and transient tranfection systems in various cell lines we tested these novel genes for their ability to block cell death in response to a variety of stimuli, including hypoxia in a neuronal cell line. Results: We found that altering the pseudosubstrate region of crmA could in some instances enhance the ability of crmA to block cell death stimuli, particularly when the region was altered to correspond to the preferred substrates of caspase 3 (CPP32) or when the p35 region was substituted into crmA. The anti-apoptosis effect of these novel genes was dependent on the cell type and the death stimulus. None of the new genes were as effective as bcl-2 in blocking hypoxia-related cell death in neuronal cells. Conclusions: Altering the pseudosubstrate region of crmA may change the ability of this gene to block cell death by altering its activity against different caspases. In response to a death stimulus such as hypoxia, blocking the downstream effectors of apoptosis may be less effective than blocking events upstream of caspase activation.