Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potentials more negative than -45 mV. When tetrodotoxin (TTX) was added to the bath, blocking INaP and leaving only an outward nonspecific cationic current (Icat), PMA had no apparent effect on responses to voltage ramps. Thus PMA did not affect Icat, and it did not induce any additional current. Intracellular application of the inactive PMA analogue, 4 alpha-PMA, did not affect INaP. The specific protein kinase C inhibitors, chelerythrine (20 microM) and calphostin C (10 microM), blocked the effect of PMA on INaP. The data suggest that PMA enhances neuronal excitability via a protein kinase C-mediated increase in INaP at functionally critical subthreshold voltages. This novel effect would modulate all neuronal functions that are influenced by INaP, including synaptic integration and active backpropagation of action potential from the soma into the dendrites.