Skeletal muscle fibres develop in cultures established from dissociated rat thymus glands. The properties of their acetylcholine receptors were compared with those on fibres in skeletal muscle cultures. Acetylcholine depolarized the thymus-derived muscle cells in a concentration-dependent manner. Under voltage clamp the reversal potential was determined to be -0.4 mV, which was not significantly different from the reversal potential of cells in the muscle cultures. In both types of cultures, sensitivity to ionophoretically applied acetylcholine decreased during the period when fibres were growing rapidly and then increased. The mean channel lifetime of acetylcholine-activated channels was similar in both types of muscle fibre (3-4 ms at -40 mV), but the single channel conductance was significantly higher in the thymus-derived cells (70-75 pS compared to 55-60 pS). The cholinoceptors on both types of muscle fibre had nicotinic properties as judged by their preferential activation by acetylcholine rather than by acetyl-beta-methylcholine and oxotremorine and by the selective blockade by tubocurarine and erabutoxin b rather than by atropine.