Characteristics of specific 125I-omega-conotoxin GVIA (125I-omega-CgTX) binding and 125I-omega-CgTX labeling using bifunctional crosslinkers were systematically investigated in crude membranes from chick whole brain. Aminoglycosides and dynorphine A (1-13) inhibited the specific binding of 125I-omega-CgTX, but not that of the L-type calcium ion channel antagonist [3H](+)PN200-110. It seems likely that the inhibitory effect of dynorphine A (1-13) does not involve kappa-opiate receptors, based on results with the opiate receptor antagonist naloxone and the kappa-opiate receptor agonist U50488H. Spider venom, Cd2+ and La3+ inhibited the specific binding of 125I-omega-CgTX, as well as that of [3H](+)PN200-110. Various L-type Ca2+ channel antagonists did not affect the specific binding of 125I-omega-CgTX. 125I-omega-CgTX specifically labeled 135 kDa and 215 kDa bands in crude membranes under reduced and non-reduced conditions, respectively. The crosslinker disuccinimidyl suberate (DSS) yielded better 125I-omega-CgTX labeling than the other two crosslinkers tested. We investigated the effect of various Ca2+ channel antagonists on 125I-omega-CgTX labeling with DSS in detail, and found that there is a strong correlation between the effects of Ca2+ channel antagonists on 125I-omega-CgTX labeling of the 135 kDa band and specific 125I-omega-CgTX binding. These results suggest that aminoglycosides and dynorphine A (1-13) are specific inhibitors of specific 125I-omega-CgTX binding, and that labeling of the 135 kDa band with 125I-omega-CgTX using DSS involves the specific binding sites of 125I-omega-CgTX, perhaps including one of the neuronal N-type Ca2+ channel subunits in the crude membranes.