Abstract: The effect of inhibition and down‐regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH‐SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31‐7549 inhibited carbachol‐evoked NA release (IC50 0.6 µM) but not 100 mM K+‐evoked release. In addition, Ro 31‐7549 inhibited the enhancement of carbachol‐ and K+‐evoked release after pretreatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 100 nM) for 8 min, with IC50 values of 0.7 and 2.4 µM, respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH‐SY5Y cells to phorbol 12,13‐dibutyrate (PDBu) or bryostatin‐1 caused down‐regulation of PKC‐α and PKC‐ε but not PKC‐ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA‐enhanced secretion was also apparent after only 2‐h exposure to either PDBu or bryostatin‐1, conditions that caused down‐regulation of PKC‐α, but not PKC‐ε or ζ. The PKC inhibitor Gö‐6976 (2 µM), which has been shown to inhibit selectively PKC‐α and β in vitro, also inhibited the TPA enhancement of carbachol‐ and K+‐evoked NA release by >50%. These data suggest that in SH‐SY5Y cells, the ability of TPA to enhance carbachol‐ and K+‐evoked NA secretion is due to activation of PKC‐\ga.