Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with alpha-bungarotoxin conjugated to tetramethylrhodamine (alpha BTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development.