The effects of nicotine, [Sarcosine]angiotensin II($[Sar^{1}]$AII; a degradation-resistant analogue of angiotensin II) and 12-0-tetradecanoylphorbol-13-acetate (TPA) on the time-course of the secretion of $[Met^{5}]$enkephalin (ME) in addition to proenkephalin A (proENK) mRNA levels were studied in bovine adrenal medullary chromaffin (BAMC) cells. Nicotine at a concentration of $10{mu}M$ caused a rapid secretion (within 1 h) of ME followed by a long-term secretion (12~24h after treatment) into the medium. Short-term stimulation of BAMC cells with $[Sar^{1}]$AII had no significant effect on secretion of ME, while long-term stimulation enhanced a profound secretion. The treatment with TPA augmented the secretion of ME 3 h after the treatment and the magnitude of increase in ME secretion induced by $[Sar^{1}]$AII or TPA continued to increase with time. Intracellular level of ME in nicotine-, $[Sar^{1}]$AII-, and TPA-treated cells were not significantly different from that of controls. In a Western blot analysis, intracellular enkephalin precursor levels in TPA- and $[Sar^{1}]$AII-treated cells were elevated, while nicotine-treated group had no effect. Long-term exposure of BAMC cells with nicotine, $[Sar^{1}]$AII and TPA also increased proENK mRNA level. Pretreatment with a calcium blocker, nimodipine ($1{mu}M$) or a calmodulin antagonist, calmidazolium ($1{mu}M$), effectively inhibited ME secretion and proENK mRNA levels induced by nicotine and $[Sar^{1}]$ All but not TPA. Pretreatment with a protein synthesis inhibitor, cycloheximide effectively inhibited ME secretion and proENK mRNA levels induced by nicotine, $[Sar^{1}]$AII or TPA. In addition, increased proenkephalin protein level induced by $[Sar^{1}]$AII but not TPA was inhibited by nimodipine. The results indicate that differential mechanisms may be involved in the regulation of ME secretion, proENK mRNA level, and processing of proenkephalin protein when BAMC cells are continuously (up to 24 h) exposed to a neuronal regulator (nicotine), hormonal regulator ($[Sar^{1}]$AII) and protein kinase C activator (TPA).