Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, we used the human colon tumor cell line HT29 (wtAPC−/−), which has been stably transfected with a zinc-inducible wild-type APC gene. The addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the β-galactosidase gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. When the e-box domain in the 5′ flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results demonstrated that wild-type APC suppressed c-myc and activated Mad1 expression in HT29 colon-derived cells. These proteins, in turn, regulated the transcription of target genes, including ODC. The data presented indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. © 2002 Wiley-Liss, Inc.