Spermidine-spermine N(1)-acetyltransferase (SSAT) is induced in response to an elevation in intracellular polyamine pools. The increased enzyme activity is the result of an increase in gene transcription, mRNA translation, and protein stability. Induction of SSAT by polyamine analogues can lead to intracellular polyamine depletion and apoptosis. The mechanism by which polyamines alter the translational efficiency of SSAT mRNA is not well understood. In this study, we investigated the regulation of SSAT translation by the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM). DENSPM induced expression of both FLAG-tagged SSAT and SSAT fused to Renilla luciferase in a time- and concentration-dependent manner. This effect was not inhibited by actinomycin D indicating that changes in gene transcription did not explain the enhanced expression in the presence of DENSPM. Furthermore, because FLAG-SSAT did not contain the 5'- or 3'-untranslated regions of SSAT, translational regulation involved the coding sequence only. By contrast, cycloheximide completely inhibited induction by DENSPM, indicating a requirement for new protein synthesis. Deletion constructs identified two regions of the SSAT protein-coding RNA sequence that conferred polyamine responsiveness. Using these regions as probes in RNA electrophoretic mobility shift assays, we observed specific binding of a cytoplasmic protein. In addition, we found that the interaction between the RNA probes and the binding protein could be inhibited by DENSPM in a concentration-dependent manner. These results suggest that polyamines regulate SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to regions of the coding sequence of the SSAT transcript.