The cholesterol concentration at the cell surface of cultured chick myocytes was increased in order to determine the effects of high levels of cholesterol on the ion channel properties of the nicotinic acetylcholine receptor. Single channel recordings and fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed under equivalent conditions for normal and cholesterol enriched myocytes. In cell attached patches from myocytes with a cholesterol to phospholipid molar ratio (c/p) of 0.24 and a microviscosity of 1.35 poise a single conductance of 51 pS was detected. The cholesterol enriched myocytes with a c/p of 0.52 and a microviscosity of 2.05 poise showed two conductances, a 54 pS and a 39 pS channel: both were blocked by alpha-bungarotoxin. The 39 pS channel was detected with the simultaneous appearance of a slow component of tau m (modulation time) for DPH fluorescence measured by phase demodulation. The 80% reduction in the open time constant (tau 2) of the 39 pS channel suggest an inhibition of the normal conformational state. The combined results suggest that cholesterol enrichment may induced a more heterogeneous lipid environment and that the two types of channel properties could result from the distribution of the receptors in different domains.