Incubation of chicken skeletal muscle fibers with an excess of anti-M-creatine kinase (CK) immunoglobulin G and an excess of anti-M-CK Fab fragments leads to heavy decoration of the M-line (Wallimann, T., D.C. Turner, and H.M. Eppenberger, 1977, J. Cell Biol. 75:297-317) and to removal of the electron-dense M-line structure (Walliman, T., G. W. Pelloni, D.C. Turner, and H.M. Eppenberger, 1978, Proc. Natl. Acad. Sci. USA., 75:4296-4300), respectively. On the other hand, incubation with low concentrations of monovalent anti-M-CK Fab did not extract but rather decorated the M-line, giving rise to a distinct two-line staining pattern. A similar double-line staining pattern, although less pronounced, was also observed within the M-line of paraformaldehyde-prefixed myogenic cells, which after permeabilization were incubated with low concentrations of divalent anti-M-CK antibody. In both cases, the two decorated lines appearing in the middle of the A-band were spaced axially 42-44 nm apart and correspond most likely to the two M4 and M4' m-bridge rows described by Sjöström and Squire (1977, J. Mol. Biol., 109:49-68; 1977, J. Microscopy., 111:239-278). It is concluded that the muscle-specific form of creatine kinase, MM-CK, contributes mainly to the electron density of these M4 and M4' m-bridges within the M-line structure. This specific labeling pattern is a further demonstration that CK is an integral part of the M-line.