The giant actin-binding protein nebulin is regarded as a component of the thin filaments in vertebrate skeletal muscles, whereas the existence of nebulin in invertebrate muscles has not yet been demonstrated. Using the cross-reactivities of polyclonal antibodies raised against nebulin from muscles of trout and lamprey. we were able to identify nebulin in the myofibrils of the cephalochordate Branchiostoma lanceolatum (lancelet) by immunoblot and immunofluorescence techniques. The similar to 720-kDa protein is localised in the I-bands of the sarcomere, where vertebrate nebulin has previously also been shown to br localised. Since lancelets have a phylogenetically key position at the vertebrate/invertebrate boundary, the detection of a high-molecular-weight nebulin indicates that nebulin-like proteins may be common to striated muscles in all chordates and increases the probability that non-chordate invertebrates also possess nebulin-related proteins.