Monoclonal antibodies reactive for B cells, T cells, T-cell subsets, killer (K) and natural killer (NK) cells, and the Ia antigen were used to analyze mononuclear cell subsets in scleroderma (SD), dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), Duchenne dystrophy (DD), and normal muscle. The analysis, which was quantitative, was performed according to diagnosis and site of accumulation. Cells at perivascular, perimysial, and endomysial sites of accumulation, and cells focally surrounding and invading nonnecrotic muscle fibers, were analyzed separately. Individual antigens were localized in 2-micron serial sections, or multiple antigens were demonstrated in a given section by sequential paired immunofluorescence. The latter approach allowed the identification of the cell phenotypes in which functional properties are defined by multiple markers, e.g., T8+ and T4+ cells that are either activated or not activated, T8+ cells that are either cytotoxic or suppressor T cells, and K/NK cells of varying maturity and killing capability. The interactions of inflammatory cells of various types with each other and the muscle fiber were further investigated by immunoelectron microscopy. In SD, the findings provide evidence for a cell-mediated immune effector response against a connective tissue and/or vascular element. In DM, the effector response appears to be predominantly humoral. In PM and IBM (but not in DM or SD), there is invasion and destruction of nonnecrotic muscle fibers by cytotoxic T cells, with or without accompanying macrophages. Because T-cell-mediated injury is antigen- and major histocompatibility complex-restricted, clones of T cells must have been sensitized previously to a muscle fiber-associated surface antigen. The identity of the putative antigen(s) remains an important, unsolved question.