How do chromaffin cell secretory stimuli program resynthesis of secreted peptides and amines? We previously showed that the physiologic nicotinic cholinergic signal for secretion also activates the biosynthesis of chromogranin A, the major protein released with catecholamines. Here, we examine signal transduction pathways whereby secretory stimuli influence exocytotic secretion versus chromogranin A transcription. Both secretion and transcription depended on initial nicotinic-triggered sodium entry into the cytosol, followed by calcium entry through -type voltage-gated channels. When calcium entered through -type channels, activation of secretion paralleled activation of transcription (r = 0.897, P = 0.002). Calcium entry from intracellular stores or through calcium ionophore channels activated secretion, though not transcription. Nicotinic-stimulated transcription depended upon protein kinase C activation; nicotine caused translocation of protein kinase C to the cell membrane fraction, and inhibition of protein kinase C blocked activation of transcription, while activation of protein kinase C mimicked nicotine effects. Transcriptional responses to both nicotine and protein kinase C mapped principally onto the chromogranin A promoter's cAMP response element (TGACGTAA; CRE box). KCREB, a dominant negative mutant of the CRE-binding protein CREB, blunted activation of chromogranin A transcription by nicotine, phorbol ester, or membrane depolarization. We conclude that activation of chromogranin A transcription by secretory stimulation in chromaffin cells is highly dependent upon precise route of calcium entry into the cytosol; transcription occurred after entry of calcium through -type channels on the cell surface, and was mediated by protein kinase C activation. The trans-acting factor CREB ultimately relays the secretory signal to the chromogranin A promoter's CRE box in cis.