Cytisine very potently binds and activates the α3β4 and α7 nicotinic subtypes, but only partially agonises the α4β2 subtype. Although with a lower affinity than cytisine, new cytisine derivatives with different substituents on the basic nitrogen (CC1–CC8) bind to both the heteromeric and homomeric subtypes, with higher affinity for brain [ 3H]epibatidine receptors. The cytisine derivatives were tested on the Ca 2+ flux of native or transfected cell lines expressing the rat α7, or human α3β4 or α4β2 subtypes using Ca 2+ dynamics in conjunction with a fluorescent image plate reader. None elicited any response at doses of up to 30–100 μM, but all inhibited agonist-induced responses. Compounds CC5 and CC7 were also electrophysiologically tested on oocyte-expressed rat α4β2, α3β4 and α7 subtypes. CC5 competitively antagonised the α4β2 and α3β4 subtypes with similar potency, whereas CC7 only partially agonised them with maximum responses of respectively 3% and 11% of those of 1 mM acetylcholine. Neither compound induced any current in the oocyte-expressed α7 subtype, and both weakly inhibited acetylcholine-induced currents. Adding chemical groups of a different class or size to the basic nitrogen of cytisine leads to compounds that lose full agonist activity on the α3β4 and α7 subtypes.