We compared excitatory synaptic transmission between hippocampal pyramidal cells in dissociated hippocampal cell cultures and in area CA3 of hippocampal slice cultures derived from wild-type mice and mice with a genetic deletion of the presynaptic growth associated protein GAP-43. The basal frequency and amplitude of action potential-dependent and -independent spontaneous excitatory postsynaptic currents were similar in both groups. The probability that any two CA3 pyramidal cells in wild-type or GAP-43 knockout (–/–) slice cultures were synaptically connected was assessed with paired recordings and was not different. Furthermore, unitary synaptic responses were similar in the two genotypes. Bath application of phorbol 12,13-diacetate (0.6–3 μm) elicited a comparable increase in the frequency of miniature excitatory synaptic currents in wild-type and GAP-43 (–/–) cultures. This effect was blocked by the protein kinase C inhibitor, bisindolylmaleimide I (1.2 μm). Finally, 3 μm phorbol 12,13-diacetate potentiated the amplitude of unitary synaptic currents to a comparable extent in wild-type and GAP-43 (–/–) slice cultures. We conclude that GAP-43 is not required for normal excitatory synaptic transmission or the potentiation of presynaptic glutamate release mediated by activation of protein kinase C in the hippocampus.