We have used the calmodulin antagonists, trifluoperazine (TFP) and calmidazolium, to study the potential role of this protein in the movement of acetylcholine receptors (AChRs) to and from the myotube membrane, as well as in the formation of clusters of AChRs within the plasma membrane. Neither calmidazolium (up to 10(-6) M) nor TFP (10(-5) M) inhibited receptor degradation or the incorporation of new receptors (12 to 24 h). In addition, neither drug blocked the increased synthesis of receptors induced by chick brain extract, nor significantly affected AChR clusters already in the plane of the membrane at the time of drug addition. However, both drugs blocked new receptor clusters (induced by a basement membrane extract from Torpedo electric organ) from forming. These results indicate that receptors can move to and from the cell membrane in a calmodulin-independent fashion, but movement in the plane of the membrane to form a cluster requires the participation of calmodulin.