The β3 neuronal nicotinic subunit is expressed in discrete regions of the brain, such as the substantia nigra, but the functional consequences of its incorporation into nicotinic receptors (nAChRs) remain unclear. We have previously shown that recombinant receptors expressed from α3, β4 and β3 subunits contain a single copy of β3 and have properties very similar to those of the background α3β4 combination (Boorman et al., 2000; 2003). Here we examine the effects of co-expressing β3 together with other nAChR subunit combinations, particularly those that are likely to be expressed in the central nervous system. Recombinant nAChRs were expressed in Xenopus oocytes by injection of excess cRNA for β3 together with cRNA for α2β2, α2β4, α3β2, α3β4, α4β2, α4β4 or α7. Currents evoked by ACh, bath-applied at a saturating concentration of 1 mM, were recorded by two-electrode voltage-clamp. Adding β3 produced an almost complete suppression of ACh responses for all combinations, except α3β4β3. Depending on the combination, the maximum response to ACh was reduced by between 88 ± 4 to 100 ± 0 % (n =5-24). We investigated the cause of this effect by using a mutated β3 subunit carrying a ‘gain-of-function’ valine to serine mutation in residue 9′ of the pore-lining region. When this mutant β3^V9′S subunit was expressed, maximum ACh responses were comparable to those obtained in the absence of β3. The properties of dose-response curves from β3^V9′S-containing receptors were consistent with the insertion of a single copy of β3. We next tested whether β3 had the same effect in rat neurones by transfecting primary hippocampal cultures (from E18 embryos, donors killed humanely following Schedule 1) with cDNA for either β3 (wild type or V9′S) or α7, together with EGFP (as a transfection marker). Under whole-cell clamp, untransfected cells (20 out of 22) showed typical α7-like responses to U-tube applications of 1-3 mM ACh. The amplitude of these responses was increased approximately ten-fold by transfection of α7 (26 out of 26 green cells). In neurones transfected with wild type β3, responses to ACh were completely suppressed (28 out of 32 cells), whereas in 47 out of 52 cells transfected with β3^V9′S, an α7-like response to ACh was detectable. In conclusion, dominant negative effects of wild-type β3 are observed in all classes of oocyte-expressed nAChRs (except α3β4) and in rat hippocampal neurons. This effect is likely to be due to a lower open probability of receptors that incorporate this subunit.