Involvement of ornithine decarboxylase (ODC) in proliferation of the HT29 cell line and its control by either fetal calf serum (FCS) or vasoactive intestinal peptide (VIP) as an external signal increasing cAMP level were investigated. Activation of the polyamine-producing system appears to be a necessary step in the proliferative response of HT29 cells since cell growth is arrested by addition of difluoromethylornithine (DFMO, an inhibitor of ODC), then restored by further addition of putrescine into the culture medium. FCS deprivation results in decreased activity of ODC and arrest of cell growth. Addition of FCS induces reactivation of ODC peaking at 9 hr and re-initiates proliferation but does not affect cAMP level. VIP strongly and rapidly stimulated cAMP accumulation, which resulted in significant activation of ODC. When VIP-induced cAMP formation was hindered by the alpha 2-adrenergic agonist UK14304, activation of ODC was no longer observed. The dose-response curve for ODC activation by VIP indicates an EC50 value of 0.078 nM which falls within the range of physiological concentrations for this peptide. However, VIP fails to stimulate proliferation when cells are cultured either in an FCS-free medium or in the presence of a growth-limiting concentration of FCS. We conclude that the mechanisms of ODC activation by either FCS or VIP are different, the latter involving cAMP formation. Activation of ODC to produce polyamines is necessary to support the proliferative process in our model but the VIP-induced activation of the enzyme alone is not sufficient to promote cell growth.