Recent work has suggested that the length of thin filaments in skeletal muscles is determined by a protein ruler, nebulin, located along the long axis of thin filaments. To examine the function of nebulin, the length distribution of the thin filaments was investigated by staining actin filaments with fluorescent rhodamine-phalloidin in rabbit cardiac muscles, which do not contain nebulin, and in skeletal muscles, which contain nebulin, by laser scanning confocal microscopy. The microscopic observation showed a difference in staining patterns between cardiac and skeletal muscle fibers when the staining was done without chemical fixation. This suggests that nebulin suppresses the attachment of phalloidin to actin filaments. Analysis of fluorescence distribution showed that the length deviation of thin filaments in the cardiac muscle was as small as that in the skeletal muscle. This indicates that the length of the thin filaments is regulated even without nebulin.