The selectivity of acetylcholine (A-Ch)-activated channels for alkali cations, organic cations, and nonelectrolytes in cultured muscle cells has been studied. To test the effect of size, charge, and hydrogen-binding capacity of permeant molecules on their permeability, we have obtained the selectivity sequences of alkali cations, compared the permeability of pairs of permeant molecules with similar size and shape but differing in charge, and studied the permeability of amines of different hydrogen bonding capacity. ACh-activated channels transport alkali cations of small hydration radii and high mobility. The molecules with positive charge and (or) a hydrogen-bond donating moiety are more permeable than the ones without. On the other hand, several nonelectrolytes, i.e., ethylene glycol, formamide, and urea, do have a small, but measurable, permeability through the channels. These results are consistent with a model that ACh-activated channel is a water-filled pore containing dipoles or hydrogen bond accepting groups and a negative charged site with a pK of 4.8.