N-type calcium channels are ω-conotoxin (ω-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the α_1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel α1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (α1B-1 and α1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The α1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal β_2 and α2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by ω-CgTx but was insensitive to dihydropyridines. The α1B-1α2bβ_2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) ω-CgTx binding sites. Co-expression of the β_2 subunit was necessary for N-type channel activity, whereas the α2b subunit appeared to modulate the expression of the channel. The heterogeneity of α1B subunits, along with the heterogeneity of α_2 and β subunits, is consistent with multiple, biophysically distinct N-type calcium channels.