Polyamines are ubiquitous molecules that are required for cell growth. Depletion of polyamines results in growth arrest or apoptosis in a cell type-dependent manner. This depletion can be achieved by induction of polyamine catabolism in response to synthetic polyamine analogues. The apoptotic response of specific tumor cell types has been directly linked to reactive oxygen species (ROS) produced by analogue-induced increases in polyamine catabolism. However, there are two polyamine catabolic pathways, and both result in the production of cytotoxic hydrogen peroxide. Currently it is not known if one pathway plays a more predominant role in determining cellular response or if different cell types respond in a cell type-specific manner. Therefore, to test directly which pathway is critical to the observed cytotoxic response of human non-small cell lung carcinoma cells, an RNA interference strategy was used to study the roles of the catabolic enzymes spermine oxidase (SMO) and spermidine/spermine N¹-acetyltransferase (SSAT) in determining drug response. These particular cells were chosen because they are the most sensitive to the toxic effects of polyamine analogue exposure. Our results indicate that SSAT-mediated polyamine depletion is the primary determinant of drug response in these cells. While SMO contributes to the analogue-mediated cytotoxic response, its activity appears to play a more minor role.