Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA-protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5'-flanking region (-1156/+13) was sufficient to confer 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cells with fresh serum-containing media rapidly induced a 5 to 6-fold increase in ODC promoter activity, maximal about 8 h after refeeding. Deletion analysis demonstrated that several sequences contribute to basal ODC promoter activity but that sequence -92/+13 was sufficient for induction by TPA or by serum. This sequence lacks canonical TPA-responsive elements, and AP-1 consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel-shift analysis of the -92/+13 sequence were competitively inhibited by wild-type but not mutant oligonucleotides encompassing a variant cyclic AMP-response element (ODC -50/-42); however a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, the proximal ODC promoter (-92/+13) responds to TPA and serum stimulation in a cell-type-specific manner that is not mediated by canonical AP-1 elements.