The effects of carbamylcholine (CCh) on the gene expression of the neuropeptide vasoactive intestinal polypeptide (VIP) were studied using two human neuroblastoma cell lines, NB-1 and BE(2)M17. CCh caused a fast increase in VIP mRNA level in both cell lines which was followed by an increase in VIP immunoreactivity. The time-course of the induction of both mRNA and peptide differed, however, between the two cell lines. No morphological changes of the cells were observed during 6 days of stimulation. The effect was mediated by the muscarinic class of acetylcholine receptors, since it could be totally abolished by atropine. Since CCh caused an accumulation of inositol-1,4,5-triphosphate, it is likely that muscarinic receptor subtype M1, M3 or M5 is involved. Experiments with the translational inhibitor, cycloheximide, showed that CCh mediated a direct effect on the VIP gene expression. By combining gel permeation chromatography with radioimmunoassays using antisera specific for the various VIP-precursor products, immunoreactive peaks eluting as the synthetic peptides were found in both cell lines. In addition, earlier eluting peaks which could represent partially processed or extended VIP forms were found. After CCh induction the concentration of all preproVIP-derived products increased, and there was a tendency towards a shift to more fully processed VIP. The findings give new evidence for a direct regulation of VIP gene expression in human neuronal cells by cholinergic agents.