Immunofluorescence and immunogold localization studies show that the two Ca²⁺‐dependent proteinases (μ‐calpain for the micromolar Ca²⁺‐requiring proteinase and m‐calpain for the millimolar Ca²⁺‐requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z‐disk of myofibrils than it is at the I‐band or A‐band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one‐tenth and one‐fifth, respectively, of the concentration at the Z‐disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m‐calpain but not μ‐calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m‐calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some μ‐and m‐calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calparns and calpastatin seem to originate from “leakage” of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.