Muscle cells in vitro and in vivo are multinucleated and express acetylcholine receptors (AcChoRs). On innervated cells, the AcChoRs form clusters which lie under the nerve terminals. However, noninnervated cells in culture also express clusters of AcChoR. Both in vivo and in vitro the AcChoR clusters appear to be associated with clusters of nuclei. We have used in situ hybridization to determine whether all the nuclei in cultured chicken embryo myotubes are equally active in expressing the AcChoR alpha subunit message. Cells were hybridized with 35S-labeled probes that contained either both an exon and an intron region or only exon sequences. Control cultures were hybridized with a labeled actin DNA probe or poly(U). The hybrids were detected by emulsion autoradiography; simultaneously, the nuclei were visualized with bisbenzamide. Cells hybridized with the intron/exon probe showed a striking preferential silver grain localization in and around some of the myotube nuclei, whereas those hybridized with the exon probe gave a rather homogeneous grain distribution in the cytoplasm. These results show that myotube nuclei possess differential activation capacities for the expression of AcChoR alpha subunit mRNA and that this difference is due to differential rates of transcription.