Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.