Recent findings suggest that protein kinase C (PKC) regulates the persistence of long-term potentiation (LTP). To test the hypothesis that PKC inhibition would decrease persistence of potentiation we applied PKC inhibitors (mellitin, polymyxin B, H-7) by micropressure ejection to the intact hippocampus either before or after LTP induction. When inhibitor was given 15 min before LTP, initial potentiation was unaffected, yet responses decayed to baseline levels by 60 min after the onset of potentiation. PKC inhibitor treatment 10 min after LTP onset induced decay of responses to pre-LTP baseline levels within 50 min of ejection. Inhibitor applied 60 min after LTP onset induced substantial decay but not to baseline levels. Potentiation was unaffected by inhibitor treatment 4 h after the induction of LTP. Measurement of PKC subcellular distribution revealed that inhibitor significantly reduced the proportion of PKC associated with the membrane. These findings represent the first demonstration that PKC inhibitors prevent persistence of potentiation. They also suggest that PKC regulates the persistence of synaptic enhancement beginning after its onset, and that PKC's role decreases with time after the induction of enhancement.