Ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase; EC 4.1.1.17) activity was increased 5-20 fold above basal activity by N(6),O(2')-dibutyryl cyclic AMP, isoproterenol, epinephrine, or fetal calf serum in confluent C6-2B rat astrocytoma cells. Serum increased ODCase activity by a mechanism apparently independent of cyclic AMP because it decreased intracellular cyclic AMP. Calcium ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) buffers (pCa = 6.4) reduced basal ODCase activity and blocked the increase in activity by beta-adrenergic agonists, dibutyryl cyclic AMP, or serum. Readdition of calcium to pCa = 4.0 restored the ability of the cells to respond to these agents. The rise in ODCase activity stimulated by isoproterenol, dibutyryl cyclic AMP, or serum was also blocked by the microtubule disrupting agents, vinblastine and colchicine, and by the microfilament disrupting agent, cytochalasin B. Lumicolchicine, an analog of colchicine that does not disrupt microtubules, was ineffective. The microtubule and microfilament disrupting agents and EGTA had no effect on the intracellular accumulation of cyclic AMP. Cycloheximide inhibited protein synthesis >95% within 30 min, and caused an immediate decline of stimulated ODCase activity with a half-time of 20-30 min. The inhibition of ODCase activity by colchicine or EGTA was distinct from that seen with cycloheximide and could not be correlated to their effects on general protein synthesis. Colchicine or EGTA each caused identical rates of decline in ODCase activity with a half-time of 20-30 min after an initial lag period of about 60 min.