Intracellular recordings were performed in voltageclamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the β3 and mutant α7 (^L247Tα7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to α-bungarotoxin but exhibited a functional profile strikingly different from that of the homomeric^L247Tα7 receptor. The heteromeric^L247T α7 β3 nAChR had a lower apparent affinity and a faster rate of desensitization than L247Tα7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type α7 (^WT α7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens ans a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of ^WTα7 and β3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that β3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.