Class II MHC antigen expression has been investigated in muscle tissue and cultured cells from normal human skeletal muscle by light and electron immunocytochemistry. In muscle tissue, these antigens were detected in satellite cells, interstitial cells, and blood vessels. In cultures, muscle cells were stained with a pan-reactive anti-HLA class II antibody and with isotypes specific for DP, DQ, and DR. The staining was present on mononucleated cells and persisted on myotubes; it was stronger for DR and DQ isotypes than for DP. At the subcellular level, staining was located not only at the cell surface, but also next to the endoplasmic reticulum and in the cytosol. Thus, myosatellite cells and aneurally cultured cells from human normal skeletal muscle express class II MHC antigens. Moreover, the myotube staining and the presence of gold particles inside the cells suggested synthesis of these antigens after myoblast fusion.