Apoptosis is now recognized as a significant problem in mammalian cell culture. Therefore, in this study, a single gene and multigene approach to inhibit apoptosis has been examined. Stable Chinese hamster ovary (CHO) cell lines were generated to overexpress different single, dual, and triple combinations of three apoptosis inhibitor genes. Two upstream inhibitors involved in the mitochondrial pathway, Bcl-X(L) and Aven, were expressed in addition to a downstream inhibitor of caspases. The caspase inhibitor, a variant of XIAP containing only the caspase inhibitory BIR domains (XIAP-BIRs), has been shown previously to enhance viabilities in mammalian cultures. Stable clonal cell lines were generated and tested for three apoptotic insults: Sindbis virus infection, the chemical reagent etoposide, and spent medium. For all single gene experiments, the Bcl-X(L)-containing cell lines provided superior protection to either the Aven- or XIAP-BIRs-containing cell lines following initial exposure to the insults. However, the cell lines expressing two or more anti-apoptosis proteins were more effective at inhibiting cell death than those expressing just one anti-apoptosis gene. The cell lines overexpressing Bcl-X(L) in combination with XIAP-BIRs were especially effective in delaying cell death for all three apoptotic insults. Expression of all three anti-apoptosis genes in concert was only slightly more effective than using Bcl-X(L) and XIAP-BIRs for some insults. During exposure to spent medium, CHO-BIRS + Aven + BclX(L) was the best inhibitor of apoptosis (IAP) initially, whereas CHO-BIRs + BclX(L) was particularly effective at later times of the experiment. In conclusion, the utilization of a mitochondrial dysfunction inhibitor used in combination with a caspase inhibitor was more effective in thwarting the progression of apoptosis than either inhibitor expressed individually. Thus, the concurrent expression of multiple apoptosis inhibitors may be the most effective strategy to increase survival of mammalian cells in culture.