1.We systematically examined the biophysical properties of ω-conotoxin GVIA-sensitive neuronal N-type channels composed of various combinations of the α(1B), α2/δ and β(1b) subunits in Xenopus oocytes. 2. Whole-cell recordings demonstrated that coexpression of the β(1b) subunit decelerated inactivation, whereas the α2/δ accelerated both activation and inactivation, and cancelled the kinetic effects of the β(1b). The α2/δ and the β(1b) controlled voltage dependence of activation differently: the β(1b) significantly shifted the current-voltage relationship towards the hyperpolarizing direction; however, the α2/δ shifted the relationship only slightly in the depolarizing direction. The extent of voltage-dependent inactivation was modified solely by the β(1b). 3. Unitary currents measured using a cell-attached patch showed stable patterns of opening that were markedly different among subunit combinations in their kinetic parameters. The α2/δ and the β(1b) subunits also acted antagonistically in regulating gating patterns of unitary N-type channels. Open time was shortened by the α2/δ, while the fraction of long opening was enhanced by the β(1b). The α2/δ decreased opening probability (P(o)), while the β(1b) increased P(o). α(1B)α2/δβ(1b) produced unitary activity with an open time distribution value in between those of α(1B)α2/δ and α(1B)β(1b). However, both the α2/δ and the β(1b) subunits reduced the number of null traces. 4. These results suggest that the auxiliary subunits alone and in combination contribute differently in forming gating apparatuses in the N-type channel, raising the possibility that subunit interaction contributes to the generation of functional diversity of N-type channels in native neuronal preparations also.