The Ca(2+)/calmodulin-activated protein phosphatase, calcineurin, is believed to regulate the development and function of skeletal and cardiac muscle. Striated muscle contains many calcineurin substrates, a few of which have been colocalized or found in molecular complexes with calcineurin. We examined the subcellular distribution of calcineurin in developing rat skeletal muscle cells and adult mouse skeletal muscle fibers by immunofluorescence microscopy. We found low levels of calcineurin immunoreactivity in the cytoplasm of myoblasts and higher levels in cytoplasmic vesicles of myotubes. Most of these vesicles were not immunoreactive for ryanodine receptors and, those that were, represented a small fraction of nascent triad junctions. In adult myofibers, calcineurin was largely associated with triads. Weaker calcineurin immunoreactivity occurred in the sarcoplasmic reticulum at the level of the M line. Unexpectedly, we found tiny clusters of calcineurin associated with nucleoli of developing myofiber nuclei. There were one to three clusters per nucleolus, either within or at the edges of fibrillar centers where ribosomal genes are transcribed. This suggests a role for calcineurin in regulating ribosome synthesis. Our findings suggest a variety of potential new targets and pathways through which calcineurin could regulate skeletal muscle development and plasticity and underscore the importance of spatial specificity in this regulation.