N-type calcium (Ca2+) channels play a critical role in synaptic function, but the mechanisms responsible for their targeting in neurons are poorly understood. N-type channels are formed by an alpha(1B) (Ca(V)2.2) pore-forming subunit associated with beta and alpha2delta auxiliary subunits. By expressing epitope-tagged recombinant alpha1B subunits in rat hippocampal neuronal cultures, we demonstrate here that synaptic targeting of N-type channels depends on neuronal contacts and synapse formation. We also establish that the C-terminal 163 aa (2177-2339) of the alpha1B-1 (Ca(V)2.2a) splice variant contain sequences that are both necessary and sufficient for synaptic targeting. By site-directed mutagenesis, we demonstrate that postsynaptic density-95/discs large/zona occludens-1 and Src homology 3 domain-binding motifs located within this region of the alpha1B subunit (Maximov et al., 1999) act as synergistic synaptic targeting signals. We also show that the recombinant modular adaptor proteins Mint1 and CASK colocalize with N-type channels in synapses. We found that the alpha1B-2 (Ca(V)2.2b) splice variant is restricted to soma and dendrites and postulated that somatodendritic and axonal/presynaptic isoforms of N-type channels are generated via alternative splicing of alpha1B C termini. These data lead us to propose that during synaptogenesis, the alpha1B-1 (Ca(V)2.2a) splice variant of the N-type Ca2+ channel pore-forming subunit is recruited to presynaptic locations by means of interactions with modular adaptor proteins Mint1 and CASK. Our results provide a novel insight into the molecular mechanisms responsible for targeting of Ca2+ channels and other synaptic proteins in neurons.