The regulation of ornithine decarboxylase (ODC) was examined in an intestinal epithelial crypt cell line (IEC-6). Addition of fetal bovine serum or growth factors to quiescent preconfluent cells resulted in a 20- to 30-fold increase in the specific activity of ODC, which was maximal at approximately 4 h. In contrast, ODC mRNA levels either did not change or increased only twofold over the time period examined. The increased enzymatic activity was blocked by cycloheximide, putrescine, and the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-napthalinesulfonamide (W-7). Cycloheximide alone increased mRNA levels and potentiated the induction in response to serum, suggesting that protein synthesis is not required for the increase in mRNA accumulation. In contrast to its effect on ODC activity, W-7 was without effect on the serum-stimulated increase in ODC or c-fos mRNA levels. Putrescine decreased ODC activity, but not mRNA content, in a dose-dependent manner with an IC50 between 0.1 and 1.0 microM. Also, serum stimulation resulted in a threefold increase in the stability of the enzyme in the presence of cycloheximide; this effect was blocked by pretreatment with W-7. Enzymatic activity was paralleled by ODC protein content as determined by [3H] difluoromethylornithine binding. Finally, the induction of enzyme activity was due entirely to an increase in Vmax as no detectable change in Km for ornithine was detected. These results suggest that ODC is regulated at multiple levels by independent signaling pathways in cultured intestinal epithelial cells. Increased levels of active ODC protein and enzymatic activity are sensitive to W-7 and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)