1. Recent studies have identified the [3H]-gabapentin-binding protein, purified from porcine cerebral cortical membranes, as the alpha 2 delta subunit of voltage-sensitive calcium channels (Gee et al., 1996). The present study investigates the influence of the polyamine spermine on specific [3H]-gabapentin binding to detergent-solubilized porcine cerebral cortical membranes. 2. Spermine, spermidine, 1,10 diaminodecane, Mg2+ and Zn2+, all divalent cations, displaced [3H]-gabapentin binding to detergent-solubilized membranes in a concentration-dependent manner with a maximal inhibition of 65-75%. Radioligand binding studies showed that spermine did not directly interact with the [3H]-gabapentin-binding site. Spermine inhibited [3H]-gabapentin binding by interacting with a polyamine-sensitive allosteric site on the membrane protein. The steep concentration-dependence of spermine inhibition of [3H]-gabapentin binding may suggest multi-site co-operativity. 3. Prolonged dialysis of cerebral cortical membranes and Tween 20-solubilized membranes resulted in a > 2.0 fold increase in [3H]-gabapentin binding. The increase in binding was due to the removal of a heat stable, low molecular weight (< 12,000Da) endogenous molecule which influences [3H]-gabapentin binding competitively. 4. Dialysis of detergent-solubilized cerebral cortical membranes also resulted in a decrease in the maximum inhibition of [3H]-gabapentin binding by spermine. Since the rates of the increase in [3H]-gabapentin binding and the loss of the ability of spermine to inhibit [3H]-gabapentin binding on dialysis were different it was inferred that a second endogenous ligand was removed during dialysis. 5. During initial steps of purification of the [3H]-gabapentin-binding protein there was a decrease in the maximum inhibition of [3H]-gabapentin binding by spermine. The loss of the second endogenous molecule during initial purification would reasonably explain the reduction in inhibition of binding by spermine. However, spermine stimulation of [3H]-gabapentin binding to material that eluted from the gel-filtration column later in the purification scheme does not appear to be due to removal of a dialysable endogenous factor or to the dissociation of other calcium channel subunit(s). 6. Adding back dialysate, before or after boiling, to detergent solubilized membranes resulted in a dose-dependent restoration of the inhibition of [3H]-gabapentin binding and of the maximal inhibition [3H]-gabapentin binding by spermine. This result is consistent with the re-addition of two endogenous heat stable ligands. 7. The findings that [3H]-gabapentin binding to the pure alpha 2 delta subunit was stimulated by spermine indicates that the alpha 2 delta subunit of voltage-sensitive calcium channels bears a modulatory spermine site. Such a spermine site has not been identified before. Spermine stimulation of [3H]-gabapentin binding to the purified protein was reversed to inhibition after adding back dialysate. Thus the inhibitory spermine effect in membranes is also probably due to one or more modulatory sites on the alpha 2 delta subunit.