Naturally-expressed nicotinic acetylcholine receptors (nAChR) containing 4 subunits (4*-nAChR) in combination with 2 subunits (42-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. 4 subunits are also richly expressed and co-localize with 4-nAChR subunits in several brain regions implicated in behavioral responses to nicotine and nicotine dependence. Thus, 44-nAChR also may exist and play important functional roles. In this study, properties were determined of human 42- and 44-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human 44-nAChR have ~4-fold higher amplitude than those mediated via human 42-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at 44-nAChR than at 42-nAChR. Cytisine and lobeline serve as full agonists at 44-nAChR but are only partial agonists at 42-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional 42- and 44-nAChR. Whole-cell current responses show stronger inward rectification for 42-nAChR than for 44-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR 2 or 4 subunits can combine with a4 subunits to generate two forms of 4*-nAChR with distinctive physiological and pharmacological features. Diversity in 4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.