The mRNAs for two key enzymes in polyamine biosynthesis, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), both have long 5′ untranslated regions (5′UTRs) that could be important in the regulation of enzyme levels by affecting the translation of these mRNAs. In order to test this hypothesis, ODC and AdoMetDC activities were measured in 3T3 cells and in 3T3 cells overexpressing eIF-4E (P2 cells). eIF-4E has been reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5′UTR. AdoMetDC activity was not greatly different in the two cell lines, but ODC activity was much greater in the P2 cells. These results were confirmed by transfecting these cells with plasmids containing a luciferase complementary DNA fused to follow the 5′UTR from ODC or AdoMetDC. The ODC 5′UTR construct produced a higher luciferase activity in the P2 cells. The high level of expression of ODC may be a critical factor in the transformed phenotype of the P2 cells since the ability of these cells to grow in soft agar was blocked by levels of the ODC inhibitor, α-difluoromethylornithine, that reduced the ODC activity to values comparable to those of the parent 3T3 cells. These results provide more evidence for a critical role of ODC activity in neoplastic transformation and for the importance of its translational regulation in cell growth and transformation.