1. We studied the effect of cotinine, a slowly eliminated metabolite of nicotine, on protein kinase C (PKC) distribution and noradrenaline release in primary cultured bovine adrenal chromaffin cells. Changes in PKC activity were detected by [3H]-phorbol-12,13-dibutyrate binding, histone phosphorylation assay and by Western blot. 2. Cotinine (10-32 mM) increased phorbol binding to chromaffin cells in response to 10 min but not to 24 h exposure. The increased binding was reversed by a nicotinic antagonist hexamethonium (10 microM). 3. Cotinine (10 mM, 30 min) also increased membrane-associated PKC activity and membrane-associated PKC alpha and epsilon immunoreactivity. 4. Cotinine (0.1-32 mM for 10 s to 20 min) dose- and time-dependently increased the release of preloaded [3H]-noradrenaline from the cultured cells. The release increased with increasing duration of the contact period. In treatments lasting 1 min or longer, a peak effect was followed by a reduced response at higher concentrations. 5. We confirm the earlier findings that cotinine is biologically active, and conclude that its effects are at least partly mediated via nicotinic cholinergic receptors and through PKC.