When a partially dehydrated muscle fibre bundle (PDM, 65% H(2)O, pH 5.5, at 4 °C) was treated with a supernatant fraction (M-line-cleaving fraction: MCF) of muscle homogenate for 5 hr, the M-lines disappeared. MCF was extracted from rabbit skeletal muscles by homogenization with 15 mM HCl containing 0.5 M NaCl (pH 3.7), fractionated with 25-65% (NH(4))(2)SO(4) and clarified by Sephadex G-75. Rabbit psoas PDM was obtained with an osmotic dehydration sheet and glycerinated. One end of the bisected fibre bundle was incubated with 10 mM Na-acetate (pH 5.5), 1 mM EDTA, 5 mM β-mercaptoethanol (β-MCE), 150 mM KCl, 10 mM NaN(3) with MCF at 25 °C for 5 hr, the muscle being stretched and relaxed several times. The other end was incubated in the same solution, except that MCF was omitted (control). Electron microscopy showed the myofibrils broken down at the M-line in the presence of MCF. The myofilaments were closely packed near the Z-line and flared out at both ends near the centre of the sarcomere (bow-tie shape). Thus, the Z-line is not the only target of proteases and structural decomposition can also occur at the M-line under specific conditions. An M-line cleaving protease may exist in the MCF muscle extract.