The effects of membrane lipid composition and fluidity on AChR ion channel function were studied after reconstituting the receptor with sphingomyelin, phosphatidylcholines with different degrees of unsaturation, or different neutral lipids. AChR ion flux activity was shown to be retained in some membranes of both high and low fluidity, as measured by the steady-state anisotropy of the membrane probes diphenylhexatriene and trimethylammonium diphenylhexatriene. The results suggest that lipid composition is more important than bulk membrane fluidity in determining AChR ion channel function.