This study examined the binding and functional properties of human α7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (−)-nicotine (100 μM) for 120 h increased the B_max values of [¹²⁵I]α-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC₅₀=970 μM) and a 6-fold upregulation was observed with the maximal concentration of (−)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (±)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [¹²⁵I]α-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the α7 nicotinic receptor antagonist, methyllycaconitine (EC₅₀=92 μM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (−)-nicotine after chronic treatment of cells with concentrations of (−)-nicotine (1000 μM) or methyllycaconitine (10 μM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505±21 pA) and 6-fold (1820±137 pA) respectively in whole cell current amplitude compared to untreated cells (267±24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of α7 nicotinic receptors and can differentially enhance nicotinic receptor function.