Growth-associated protein 43 (GAP-43) and neurogranin are protein kinase C substrate proteins that are thought to play an important role in synaptic plasticity, but little is currently known about the mechanisms that may regulate their function at the synapse. In this study, we show that long-term elevation of intracellular cAMP levels in rat primary cortical cultures results in a persistent downregulation of GAP-43 and neurogranin, most likely at the transcriptional level. This effect may be at least partially mediated by protein kinase A, but is independent of protein kinase C activation. Moreover, it is mimicked and occluded by manipulations that alter the levels of spontaneous synaptic activity in primary cultures, such as bicuculline and tetrodotoxin. These data suggest that levels of GAP-43 and neurogranin are regulated by factors known to modulate synaptic strength, thus providing a potential mechanism by which protein kinase C signaling pathways and their substrates might contribute to synaptic function and/or plasticity.