Excitatory synaptic transmission in the hippocampus involves the participation of at least two types of presynaptic Ca2+ channels, N- type channels sensitive to omega-conotoxin GVIA (omega-CTx GVIA) and Q- type channels sensitive to omega-agatoxin IVA (omega-Aga IVA). Hippocampal pyramidal neurons in cell culture were used to examine the participation of these two classes of channels at different stages of synapse development. Specific Ca2+ channel toxins were used to block presynaptic Ca2+ channels while whole-cell voltage-clamp recordings were used to record evoked EPSCs in postsynaptic neurons. At immature synapses (cells in culture for 10–15 d), omega-CTx GVIA (1–5 microM) blocked transmission by more than 80% while omega-Aga IVA (1 microM) was less effective. In older cultures, however, omega-Aga IVA (1 microM) was more effective than omega-CTx GVIA (1–5 microM) in blocking synaptic transmission. The pharmacological properties of the omega-Aga IVA sensitive component of synaptic transmission were examined in more detail using omega-Aga IVA and omega-conotoxin MVIIC (omega-CTx MVIIC). The properties of this component of transmitter release indicated that a Q-type Ca2+ channel was involved in presynaptic Ca2+ entry. The results suggest that different classes of presynaptic Ca2+ channels begin to participate in transmitter release at different times during synapse development and maturation.