Using perforated-patch recordings, we have examined the part played by endogenous G-protein subunits in the α2-adrenoceptor-mediated inhibition of N-type Ca2+ currents in sympathetic neurones.Two components of ICa inhibition by noradrenaline were recorded: a prominent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTX-resistant pathway.PTX-sensitive inhibition was reduced by microinjection of antibodies against either GαoA,B or Gαi1,2. The voltage-dependent fraction of inhibition was reduced by anti-Gαo but not by anti-Gαi antibody.Antisense depletion of GαoA led to a marked reduction of noradrenaline-induced inhibition and voltage dependence. By contrast, Gαi depletion attenuated noradrenergic modulation without affecting the voltage dependence.Expression of the βγ-binding agents β-adrenergic receptor kinase 1 (C-terminus, βARK1C-ter) or Gαi1 with a Cys3 to Ser mutation partially prevented noradrenergic inhibition while α-transducin abolished it. Residual inhibition was mostly voltage independent in cells expressing βARK1C-ter but was strongly reversed by depolarization in Gαi1 Cys3Ser-expressing cells.Expression of the PTX-resistant Gαi1 Cys351Ile mutant in cells treated with PTX restored α2-adrenoceptor inhibition. This restored inhibition was weakly reversed by depolarization. Both the degree and voltage dependence of inhibition were correlated with the level of expression of the Gαi1 Cys351Ile subunit.Our findings identify βγ dimers associated with GαoA and Gαi as mediators of the PTX-sensitive α2-adrenoceptor-mediated inhibition of N-type Ca2+ channels. Different βγ combinations may account for the differential voltage-dependent effects of Go and Gi on ICa.