The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF: is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.