The human neuroblastoma cell line SH-SY5Y is a well characterized model for sympathetic neuronal differentiation in vitro. Several differentiation protocols exist, one of which, the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum, has been thoroughly studied. Wild-type SH-SY5Y cells are unresponsive to nerve growth factor (NGF), but cells transfected with the high-affinity NGF receptor TrkA (SH-SY5Y/TrkA) differentiate in response to NGF. In the present study, we have addressed the existence of a differentiation-specific mode of activation and subcellular distribution of the extracellular signal-regulated kinases ERK1 and ERK2 in SH-SY5Y/wt and SH-SY5Y/TrkA. Both TPA and NGF induced a sustained activation and nuclear accumulation of ERK that was accompanied by transactivation of a serum response element (SRE)-driven reporter and of the c-fos gene. However, activation and nuclear accumulation of ERK were not sufficient to induce neuronal differentiation in SH-SY5Y, as demonstrated by the response to TPA in serum-free cultures. Nuclear accumulation but not activation of ERK was demonstrated to require active protein kinase C (PKC). The effect of specific PKC inhibitors on subcellular distribution of ERK and ERK-dependent transcription suggests a functional role for PKC in the regulation of nuclear ERK activity in SH-SY5Y neuroblastoma cells.